Authentic trimeric HIV-1 GP140 envelope glycoproteins comprising a long linker and tag

ABSTRACT

An approach of producing recombinant trimers that mimic native HIV-1 envelope trimers is developed. A recombinant protein forming the recombinant trimers encompasses a recombinant HIV-1 gp140 fused to a tag through a linker at C-terminus of the recombinant HIV-1 gp140. The linker is sufficiently long so that the tag is accessible for binding by a binding molecule bound on a solid matrix. After expressed in a cell, the recombinant protein is secreted into the culture medium and assembles into recombinant trimers therein. The recombinant trimers may be directly purified from the culture medium. Cleaved and uncleaved trimers from different clade viruses are produced.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. Provisional PatentApplication No. 62/133,578, entitled “A NEW APPROACH TO PRODUCE HIV-1GP140 ENVELOPE PROTEIN TRIMERS,” filed Mar. 16, 2015; U.S. ProvisionalPatent Application No. 62/166,271, entitled “A NEW APPROACH TO PRODUCEHIV-1 ENVELOPE TRIMERS: BOTH CLEAVAGE AND PROPER GLYCOSYLATION AREESSENTIAL TO GENERATE AUTHENTIC TRIMERS,” filed May 26, 2015. The entirecontents and disclosures of these provisional patent applications areincorporated herein by reference.

This application makes reference to U.S. Provisional Patent ApplicationNo. 61/731,147, entitled “DESIGNING A SOLUBLE FULL-LENGTH HIV-1 GP41TRIMER,” filed Nov. 29, 2012 and U.S. patent application Ser. No.14/091,401, entitled “DESIGNING A SOLUBLE FULL-LENGTH HIV-1 GP41TRIMER,” filed Nov. 27, 2013. The entire disclosure and contents ofthese patent applications are incorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention is made with government support under NIH grant AI102725awarded by National Institute of Allergy and Infectious Diseases (NIAID)to Venigalla B. Rao. The U.S. Government has certain rights in thisinvention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 12, 2017, isnamed 109007-420004_SL.txt and is 25,153 bytes in size.

BACKGROUND Field of the Invention

The present invention relates to HIV-1 envelope protein gp140 and HIV-1envelope trimers, as well as an HIV vaccine.

Related Art

The trimeric envelope glycoprotein spike of HIV-1 mediates virus entryinto human cells. The exposed part of the trimer, gp140, consists of twononcovalently associated subunits, gp120 and gp41 ectodomain. Thesurface of an HIV-1 virus is covered with 20-50 trimeric envelope spikesthat are embedded in the viral envelope. The spike makes the firstcontact with the human T cell during sexual transmission of the virus.This interaction triggers a series of events leading to fusion of viraland host membranes and delivery of the virus nucleocapsid core into thehost cell. This results in the establishment of HIV infection in thehuman host. A vaccine containing recombinantly produced viral trimersthat mimics native spike might elicit trimer-specific antibodies, whichby binding to the virus can disable envelope protein (Env) function andblock the transmission of HIV into humans. Therefore, development of arecombinant trimer immunogen as a vaccine has been one of the toppriorities in the hunt for an effective HIV vaccine. However,preparation of authentic HIV-1 trimers has been challenging. Theprocedures developed so far have not produced authentic trimers, whichappear as three-blade propeller shaped particles when visualized by anelectron microscope.

SUMMARY

According to a first broad aspect, the present invention provides arecombinant protein comprising: a recombinant HIV-1 gp140, a linker, anda tag, wherein the tag is fused to C-terminus of the recombinant HIV-1gp140 through the linker, wherein the linker is sufficiently long sothat the tag is accessible for binding by a binding molecule bound on asolid matrix, and wherein the recombinant protein assembles intorecombinant trimers that mimic native HIV-1 envelope trimers in culturemedium when the recombinant protein is produced by cells growing in theculture medium.

According to a second broad aspect, the present invention provides acomposition comprising a recombinant trimer of a heterodimer comprisinga cleaved gp120 and a cleaved gp41 ectodomain, wherein the cleaved gp120and the cleaved gp41 ectodomain are covalently associated through adisulfide bond, wherein the cleaved gp41 ectodomain is fused to a tagthrough a linker at C-terminus of the gp41 ectodomain, wherein thelinker is sufficiently long so that the tag is accessible for binding bya binding molecule bound on a solid matrix, and wherein the recombinanttrimer mimics a native HIV-1 envelope trimer.

According to a third broad aspect, the present invention provides acomposition comprising a recombinant trimer of a recombinant protein,wherein the recombinant protein comprises a recombinant HIV-1 gp140fused to a tag through a linker at C-terminus of the recombinant HIV-1gp140, wherein the linker is sufficiently long so that the tag isaccessible for binding by a binding molecule bound on a solid matrix,and wherein the recombinant trimer mimics a native HIV-1 envelopetrimer.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and constitutepart of this specification, illustrate exemplary embodiments of theinvention, and, together with the general description given above andthe detailed description given below, serve to explain the features ofthe invention.

FIG. 1 is a schematic diagram showing a recombinant trimer of envelopeprotein gp140 fused to tags through long linkers according to oneembodiment of the present invention in comparison with a recombinanttrimer of envelope protein gp140 fused to tags without long linkers.

FIG. 2 is a set of schematic images of gp140 expression cassetteencompassing various exemplary linkers and tags attached to an HIV-1gp140 C-terminus according to some embodiments of the present invention.Figure discloses SEQ ID NOS 18-23, respectively, in order of appearanceand “Octa-His tag” as SEQ ID NO: 3.

FIG. 3 is a schematic drawing of a plasmid vector comprising expressioncassette according to some embodiments of the present invention.

FIG. 4 is a schematic drawing of a JRFL foldon-gp140 recombinantconstruct showing the positions of the cleavage resistant SEKS mutation(SEQ ID NO: 15), foldon, and His-tag according to some embodiments ofthe present invention. Figure discloses “6× His” as SEQ ID NO: 4,respectively, in order of appearance.

FIG. 5 is a graph of elution profile of trimers and oligomers on SEC forthe purification of uncleaved JRFL gp140 foldon trimers according tosome embodiments of the present invention.

FIG. 6 is an image of native gel of starting material from HisTrapcolumn that is loaded on SEC (lane S) according to some embodiments ofthe present invention.

FIG. 7 is an image of native gel of the SEC fractions according to someembodiments of the present invention.

FIG. 8 is an image of negative-stain EM of the peak trimer fraction fromFIG. 4 according to some embodiments of the present invention.

FIG. 9 is an image of 2D class averages of foldon trimers from FIG. 7according to some embodiments of the present invention.

FIG. 10 is an image of reducing SDS polyacrylamide gel showing proteinpatterns of the samples as indicated at the top according to oneembodiment of the present invention.

FIG. 11 is a set of images of reducing SDS polyacrylamide gels showingprotein patterns of the samples as indicated at the top according to oneembodiment of the present invention.

FIG. 12 is an image of Blue native (BN) gel of STREP-TACTIN® purifiedgp140 samples according to one embodiment of the present invention.

FIG. 13 is an image of SDS gel of samples under reducing (+DTT) ornon-reducing (−DTT) conditions according to one embodiment of thepresent invention.

FIG. 14 is an image of Western blot using STREP-TAG® II specific mAbaccording to one embodiment of the present invention.

FIG. 15 is a schematic illustration of an example of screening strategyto optimize recombinant gp140 production according to one embodiment ofthe present invention according to one embodiment of the presentinvention.

FIG. 16 is a set of images of non-reducing SDS gel comparing theproduction of uncleaved and cleaved gp140 in the culture medium foramino acid truncations at aa664 and aa683 according to one embodiment ofthe present invention.

FIG. 17 is an image showing reducing SDS gel comparing the production ofgp140 recombinants truncated at various amino acid positions at theC-terminus according to one embodiment of the present invention.

FIG. 18 is an image showing reducing SDS gel of uncleaved and cleavedgp140 proteins truncated at aa664 and aa683 according to one embodimentof the present invention.

FIG. 19 is an image showing BN gel of STREP-TACTIN® purified gp140samples according to one embodiment of the present invention.

FIG. 20 is an image of reducing SDS gel of various gp140 samples showingsingle-step purification of Strep-Tagged gp140 from the culturesupernatant by STREP-TACTIN® column according to one embodiment of thepresent invention according to one embodiment of the present invention.

FIG. 21 is an image showing BN gel of STREP-TACTIN® purified gp140 thatis loaded on SEC (lane S) and three major fractions eluted from SEC(lanes 1-3 corresponding to the pooled peaks 1-3 shown in FIG. 21)according to one embodiment of the present invention.

FIG. 22 is an image showing a typical elution profile of gp140 oligomersfrom Superdex 200 size exclusion column (SEC) according to oneembodiment of the present invention according to one embodiment of thepresent invention.

FIG. 23 is an image of BN gel showing purification of cleaved trimersexpressed in 293F cells according to one embodiment of the presentinvention.

FIG. 24 is an image of reducing SDS gel of fractions showingpurification of cleaved trimers expressed in 293F cells according to oneembodiment of the present invention.

FIG. 25 is an image of negative-stain EM of the peak SEC fraction ofpurification of cleaved trimers expressed in 293F cells according to oneembodiment of the present invention.

FIG. 26 is an image of negative-stain EM of the peak SEC fractionpurification of cleaved trimers expressed in 293F cells according to oneembodiment of the present invention.

FIG. 27 is an image of reference-free 2D class averages of cleavedtrimers expressed in 293F cells according to one embodiment of thepresent invention.

FIG. 28 is an image of BN gel showing purification of cleaved trimersexpressed in GnTI⁻ cells according to one embodiment of the presentinvention.

FIG. 29 is an image of reducing SDS gel of fractions showingpurification of cleaved trimers expressed in GnTI⁻ cells according toone embodiment of the present invention.

FIG. 30 is an image of negative-stain EM of the peak SEC fraction ofpurification of cleaved trimers expressed in GnTI⁻ cells according toone embodiment of the present invention.

FIG. 31 is an image of negative-stain EM of the peak SEC fraction ofpurification of cleaved trimers expressed in GnTI⁻ cells according toone embodiment of the present invention.

FIG. 32 is an image of reference-free 2D class averages of purifiedcleaved trimers expressed in GNTF cells according to one embodiment ofthe present invention.

FIG. 33 is an image of BN gel showing purification of uncleaved trimersexpressed in 293F cells according to one embodiment of the presentinvention.

FIG. 34 is an image of reducing SDS gel of fractions showingpurification of uncleaved trimers expressed in 293F cells according toone embodiment of the present invention.

FIG. 35 is an image of negative-stain EM of the peak SEC fraction ofpurification of uncleaved trimers expressed in 293F cells according toone embodiment of the present invention.

FIG. 36 is an image of negative-stain EM of the peak SEC fractionpurification of uncleaved trimers expressed in 293F cells according toone embodiment of the present invention.

FIG. 37 is an image of negative-stain EM of the peak SEC fractionpurification of uncleaved trimers expressed in 293F cells according toone embodiment of the present invention.

FIG. 38 is an image of BN gel showing purification of uncleaved trimersexpressed in GnTI⁻ cells according to one embodiment of the presentinvention.

FIG. 39 is an image of reducing SDS gel of fractions showingpurification of uncleaved trimers expressed in GnTI⁻ cells according toone embodiment of the present invention.

FIG. 40 is an image of negative-stain EM of the peak SEC fraction ofpurification of uncleaved trimers expressed in GnTI⁻ cells according toone embodiment of the present invention.

FIG. 41 is an image of negative-stain EM of the peak SEC fractionpurification of uncleaved trimers expressed in GnTI⁻ cells according toone embodiment of the present invention.

FIG. 42 is an image of negative-stain EM of the peak SEC fractionpurification of uncleaved trimers expressed in GnTI⁻ cells according toone embodiment of the present invention.

FIG. 43 is a graph illustrating conformational heterogeneity ofuncleaved trimers produced in 293F cells according to one embodiment ofthe present invention.

FIG. 44 is a set of gel images showing that protomers of uncleavedtrimers are nonspecifically crosslinked with disulfide bonds accordingto one embodiment of the present invention.

FIG. 45 is a set of gel image showing Proteinase K sensitivity ofcleaved and uncleaved JRFL trimers according to one embodiment of thepresent invention.

FIG. 46 is an image of reducing SDS gel showing the ladder of gp41ectodomain bands according to one embodiment of the present invention.

FIG. 47 is an image shows densitometric quantification of the intensityof the gp41 ladder bands shown in FIG. 46 according to one embodiment ofthe present invention.

FIG. 48 is an image of a Western blot of reducing SDS gel usingSTREP-TAG® II specific mAb according to one embodiment of the presentinvention.

FIG. 49 is an image of Coomassie blue-stained non-reducing SDS gel ofpurified uncleaved (CR) and cleaved (CP) JRFL and BG505 gp140 trimersproduced in 293F or GnTI⁻ cells according to one embodiment of thepresent invention.

FIG. 50 is an image of Coomassie blue-stained non-reducing SDS gel ofsamples from FIG. 49 after treatment with PNGase F according to oneembodiment of the present invention.

FIG. 51 is an image showing the epitope signatures recognized by variousantibodies (Abs) in the 3D context of a gp140 trimer structure (PDB4tvp).

FIG. 52 is a set of graphs of results of ELISA performed with purifiedcleaved and uncleaved gp140 trimers with various mAbs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

Where the definition of terms departs from the commonly used meaning ofthe term, applicant intends to utilize the definitions provided below,unless specifically indicated.

For purposes of the present invention, it should be noted that thesingular forms, “a,” “an” and “the” include reference to the pluralunless the context as herein presented clearly indicates otherwise.

For purposes of the present invention, directional terms such as “top,”“bottom,” “upper,” “lower,” “above,” “below,” “left,” “right,”“horizontal,” “vertical,” “up,” “down,” etc., are used merely forconvenience in describing the various embodiments of the presentinvention. The embodiments of the present invention may be oriented invarious ways. For example, the diagrams, apparatuses, etc., shown in thedrawing figures may be flipped over, rotated by 90° in any direction,reversed, etc.

For purposes of the present invention, the term “comprising”, the term“having”, and the term “including” are intended to be open-ended andmean that there may be additional elements other than the listedelements.

For purposes of the present invention, the term “access” refers toapproaching near and being able to contact or interact with a molecule.For example, when a binding molecule accesses a target peptide, thebinding molecule approaches near the target peptide and then binds tothe target peptide. A binding molecule that is accessible to a targetpeptide is not blocked or sterically hindered by the 3-dimensional (3-D)structure of a protein or a protein complex comprising the targetpeptide. For example, if a peptide tag fused at an end of a protein or aprotein complex is accessible for binding by a binding molecule bound ona solid matrix, the access and binding of the binding molecule bound ona solid matrix to the peptide tag is not sterically hindered by the 3-Dstructure of the protein or the protein complex.

For purposes of the present invention, the term “accessible for binding”refers to a binding site of a target molecule or target complex that isnot blocked or sterically hindered by the 3-D structure of the targetmolecule or target complex with respect to a binding molecule binding tothe binding site. For example, in one embodiment of the presentinvention where the binding site is a tag fused to a gp140 protein, along linker is used to separate the tag from the gp140 protein to makethe tag available to be bound by a binding molecule, thereby preventingstructures of the gp140 protein from sterically hindering or blockingaccess of the binding molecule to the tag.

For purposes of the present invention, the term “affinitychromatography” refers to a separation technique based upon molecularconformation, which frequently utilizes application specific resins.These resins have ligands attached to their surfaces which are specificfor the compounds to be separated. Most frequently, these ligandsfunction in a fashion similar to that of antibody-antigen interactions.For example, many membrane proteins are glycoproteins and can bepurified by lectin affinity chromatography. Detergent-solubilizedproteins can be allowed to bind to a chromatography resin that has beenmodified to have a covalently attached lectin. Proteins that do not bindto the lectin are washed away and then specifically bound glycoproteinscan be eluted by adding a high concentration of a sugar that competeswith the bound glycoproteins at the lectin binding site. Some lectinshave high affinity binding to oligosaccharides of glycoproteins that ishard to compete with sugars, and bound glycoproteins need to be releasedby denaturing the lectin.

For purposes of the present invention, the term “antibody” refers to aprotein produced by plasma cells that are used by an immune system toidentify and neutralize foreign objects, for example, bacteria andviruses. An “antibody” is also known as an “immunoglobulin.” Eachantibody recognizes a specific part of a specific foreign object, calledan antigen, and binds the specific antigen. Antibodies can causeagglutination and precipitation of antibody-antigen products, prime forphagocytosis by macrophages and other cells, block viral receptors, andstimulate other immune responses, such as the complement pathway.

For purposes of the present invention, the term “attach,” when is usedin protein or polypeptide, refers to join, fuse, link, or connect twoamino acid sequences together. For example, an amino acid tag may beattached to a polypeptide of interest such as a gp140 at C-terminus orN-terminus. Usually, a tag is attached or fused to a polypeptide througha linker, wherein the linker locates between the polypeptide and thetag.

For purposes of the present invention, the term “base of a trimer” andthe term “base of a gp140 structure” are used interchangeably to referto the portion of a gp140 subunit within the trimer or the gp140structure not including other molecules bound, attached or complexedwith one of the gp140 subunits of the trimer. For example, linkers,tags, etc. are not part of the base of a trimer or the base of a gp140structure.

For purposes of the present invention, the term “bind,” the term“binding” or the term “bound” refers to any type of chemical or physicalbinding, which includes but is not limited to covalent binding, hydrogenbinding, electrostatic binding, biological tethers, transmembraneattachment, cell surface attachment and expression.

For purposes of the present invention, the term “binding molecule”refers to a molecule having a specific interaction with a target complexsuch as a DNA, a protein, a polypeptide, or a polypeptide oligomer, etc.A specific interaction between the binding molecule and the targetcomplex may be a highly specific interaction such as an interactionbetween antigen and antibody, or receptor and ligand. A binding moleculemay be bound to a solid matrix such as agarose beads and then be used inprotein affinity purification to capture a target complex from a mixturecontaining the target complex. A binding molecule bound to a solidmatrix may be a Ni-NTA bead, a STREP-TACTIN® bead, etc. For example, inaffinity purification of a protein or an antigen from a mixture, anantibody against a target protein or a target antigen may be used as abinding molecule and be bound to a bead to capture the protein or theantigen from the mixture. In some examples, a binding molecule may be asmall molecule specifically binds to a tag fused on a recombinantprotein.

For purposes of the present invention, the term “clade” refers torelated human immunodeficiency viruses (HIVs) classified according totheir degree of genetic similarity. There are currently three groups ofHIV-1 isolates: M, N and O. Group M (major strains) consists of at leastten clades, A through J. Group O (outer strains) may consist of asimilar number of clades. Group N is a new HIV-1 isolate that has notbeen categorized in either group M or O. In certain exemplaryembodiments, a composition of the invention (e.g., any one of thevaccines of the first or fourth aspects, the compositions of the thirdaspect, the nucleic acid molecules of the fifth aspect, and/or thevectors of the sixth aspect) as described herein will recognize andraise an immune response (e.g., neutralizing anti-HIV antisera) againsttwo, three, four, five, six, seven, eight, nine, ten or more cladesand/or two or more groups of HIV.

For purposes of the present invention, the term “cleavage” refers tobreaking of a chemical bond in a polypeptide molecule to separate ordivide a polypeptide molecule into two or more portions such as twosmall peptides.

For purposes of the present invention, the term “correspond” and theterm “corresponding” refer to that a protein sequence referinterchangeably to an amino acid position(s) of a protein. An amino acidat a position of a protein may be found to be equivalent orcorresponding to an amino acid at a position of one or more otherprotein(s) based on any relevant evidence, such as the primary sequencecontext of the each amino acid, its position in relation to theN-terminal and C-terminal ends of its respective protein, the structuraland functional roles of each amino acid in its respective protein, etc.

For purposes of the present invention, the term “constitutively express”refers to the consistent synthesis of a protein. “Constitutivelyexpress” is contrary to “inducible expression” which depends onpromoters that respond to the induction conditions.

For purposes of the present invention, the term “crosslink” refers to abond that links one polypeptide to another. Proteins naturally presentin the body can contain crosslinks generated by enzyme-catalyzed orspontaneous reactions. Such crosslinks are important in generatingmechanically stable structures such as hair, skin and cartilage.Disulfide bond formation is one of the most common crosslinks, butisopeptide bond formation is also common. Proteins can also becross-linked artificially using small-molecule crosslinkers. Compromisedcollagen in the cornea, a condition known as keratoconus, can be treatedwith clinical crosslinking.

For purposes of the present invention, the term “culture medium,” theterm “culture supernatant,” and the term “cell culture supernatant”refer to the media/fluid in which cells are suspended/cultured duringgrowth. Culture supernatant is usually the clear upper liquid part of amixture including cells and media after being centrifuged. Culturesupernatant may also be the liquid lying above a layer of precipitatedcells.

For purposes of the present invention, the term “domain” and the term“protein domain” refer to a distinct functional or structural unit in aprotein. Usually, a protein domain is responsible for a particularfunction or interaction, contributing to the overall role of a protein.Domains may exist in a variety of biological contexts, where similardomains can be found in proteins with different functions.

For purposes of the present invention, the term “ectodomain” refers to adomain of a membrane protein that extends into the extracellular space(e.g., a space outside a cell). Ectodomains are usually the parts ofproteins that initiate contact with surfaces, which leads to signaltransduction. For example, the ectodomain of an HIV-1 envelopeglycoprotein (Env) is a heterodimer with mass of approximately 140 kDa,composed of the entire gp120 component, and approximately 20 kDa ofgp41, which are displayed on the surface of the viral membrane.

For purposes of the present invention, the term “engineered” refers tobeing made by biological engineering.

For purposes of the present invention, the term “Env spike” refers to astructure of a complex existing on the envelope of an HIV viralparticle. An HIV Env spike is a trimer formed by envelope protein Envgp140.

For purposes of the present invention, the term “envelope glycoprotein”and the term “Env” refer to, but are not limited to, the glycoproteinthat is expressed on the surface of the envelope of HIV virions and thesurface of the plasma membrane of HIV infected cells. For example, anative env gene encodes gp160, which is proteolytically cleaved into thegp120 and gp41 Envelope (Env) proteins. Gp120 binds to the CD4 receptoron a target cell that has such a receptor, such as, e.g., a T-helpercell. A native Gp41 is non-covalently bound to gp120, and provides thesecond step by which HIV enters the cell. It is originally buried withinthe viral envelope, but when gp120 binds to a CD4 receptor, gp120changes its conformation causing gp41 to become exposed, where it canassist in fusion with the host cell.

For purposes of the present invention, the term “epitope” refers to amolecular region on the surface of an antigen capable of eliciting animmune response and combining with the specific antibody produced bysuch a response. It is also called “antigenic determinant.” T cellepitopes are presented on the surface of an antigen-presenting cell,where they are bound to MHC molecules.

For purposes of the present invention, the term “expression” and theterm “gene expression” refer to a process by which information from agene or a fragment of DNA is used in the synthesis of a functional geneproduct. A gene which encodes a protein will, when expressed, betranscribed and translated to produce that protein.

For purposes of the present invention, the term “expression cassette”refers to a part of a vector DNA used for cloning and transformation. Ineach successful transformation, the expression cassette directs thecell's machinery to make RNA and protein. Some expression cassettes aredesigned for modular cloning of protein-encoding sequences so that thesame cassette can easily be altered to make different proteins.Expression cassettes may also refer to a recombinantly produced nucleicacid molecule that is capable of expressing a genetic sequence in acell. An expression cassette typically includes a regulatory region suchas a promoter, (allowing transcription initiation), and a sequenceencoding one or more proteins or RNAs. Optionally, the expressioncassette may include transcriptional enhancers, non-coding sequences,splicing signals, transcription termination signals, and polyadenylationsignals. The sequences controlling the expression of the gene, i.e. itstranscription and the translation of the transcription product, arecommonly referred to as regulatory unit. Most parts of the regulatoryunit are located upstream of coding sequence of the heterologous geneand are operably linked thereto. The expression cassette may alsocontain a downstream 3′ untranslated region comprising a polyadenylationsite. The regulatory unit of the invention is either directly linked tothe gene to be expressed, i.e. transcription unit, or is separatedtherefrom by intervening DNA such as for example by the 5′-untranslatedregion of the heterologous gene. Preferably the expression cassette isflanked by one or more suitable restriction sites in order to enable theinsertion of the expression cassette into a vector and/or its excisionfrom a vector. Thus, the expression cassette according to the presentinvention can be used for the construction of an expression vector, inparticular a mammalian expression vector.

For purposes of the present invention, the term “expression vector,”otherwise known as an expression construct, refers to a plasmid or virusdesigned for protein expression in cells. The vector is used tointroduce a specific gene into a target cell, and can commandeer thecell's mechanism for protein synthesis to produce the protein encoded bythe gene. The plasmid is engineered to contain regulatory sequences thatact as enhancer and promoter regions and lead to efficient transcriptionof the gene carried on the expression vector. The goal of awell-designed expression vector is the production of significant amountof stable messenger RNA, and therefore proteins.

For purposes of the present invention, the term “foldon sequence” refersto a sequence derived from the native T4 phage fibritin. The foldonsequence has a sequence of GYIPEAPRDGQAYVRKDG EWVLLSTFL (SEQ ID NO: 1).When incorporated at the C-terminal of a protein molecule, the foldonsequence stabilizes the triple helix formed in the protein.

For purposes of the present invention, the term “fragment” of a moleculesuch as a protein or a nucleic acid refers to a portion of an amino acidsequence of the protein or a portion of a nucleotide sequence of thenucleic acid.

For purposes of the present invention, the term “furin” refers to aprotein encoded by the FURIN gene. Some proteins are inactive when theyare first synthesized, and must have sections deleted in order to becomeactive. Furin deletes these sections and activates the proteins. Furinis one of the proteases responsible for the proteolytic cleavage of HIVenvelope polyprotein precursor gp160 to gp120 and gp41 prior to viralassembly.

For purposes of the present invention, the term “furin cleavageproficient (CP)” refers to offering proficiency for cleavage by furin. Aprotein with furin cleavage proficient site is likely to be cleaved byfurin.

For purposes of the present invention, the term “furin cleavageresistant (CR)” refers to offering resistance to be cleaved by furin. Aprotein with furin cleavage resistant site is unlikely to be cleaved byfurin.

For purposes of the present invention, the term “fuse” refers to jointogether physically, or to make things join together and become a singlething.

For purposes of the present invention, the term “fusion polypeptide” orthe term “fusion protein” refers to a protein having at least twoheterologous polypeptides covalently linked, either directly or via anamino acid linker. The heterologous polypeptides forming a fusionprotein are typically linked C-terminus to N-terminus, although they canalso be linked C-terminus to C-terminus, N-terminus to N-terminus, orN-terminus to C-terminus. The polypeptides of the fusion protein can bein any order and may include more than one of either or both of theconstituent polypeptides. These terms encompass conservatively modifiedvariants, polymorphic variants, alleles, mutants, subsequences,interspecies homologs, and immunogenic fragments of the antigens thatmake up the fusion protein. These terms may also refer to a proteindeveloped from a fusion gene that is created through a joining of two ormore genes originally coding for separate proteins. Translation of thisfusion gene results in a single or multiple polypeptides with functionalproperties derived from each of the original proteins. In presentinvention, “fusion protein” and “recombinant protein” areinterchangeable. Fusion proteins of the disclosure may also compriseadditional copies of a component antigen or immunogenic fragmentthereof.

For purposes of the present invention, the term “fusion polypeptide” orthe term “fusion protein” refers to a protein having at least twoheterologous polypeptides covalently linked, either directly or via anamino acid linker. The heterologous polypeptides forming a fusionprotein are typically linked C-terminus to N-terminus, although they canalso be linked C-terminus to C-terminus, N-terminus to N-terminus, orN-terminus to C-terminus. The polypeptides of the fusion protein can bein any order and may include more than one of either or both of theconstituent polypeptides. These terms encompass conservatively modifiedvariants, polymorphic variants, alleles, mutants, subsequences,interspecies homologs, and immunogenic fragments of the antigens thatmake up the fusion protein. These terms may also refer to a proteindeveloped from a fusion gene that is created through a joining of two ormore genes originally coding for separate proteins. Translation of thisfusion gene results in a single or multiple polypeptides with functionalproperties derived from each of the original proteins. In presentinvention, “fusion protein” and “recombinant protein” areinterchangeable. Fusion proteins of the disclosure may also compriseadditional copies of a component antigen or immunogenic fragmentthereof.

For purposes of the present invention, the term “gel electrophoresis”refers to a method for separation and analysis of macromolecules (DNA,RNA and proteins) and their fragments, based on their size and charge.Gel electrophoresis conditions include denaturing condition and nativecondition (non-denaturing condition). Under denaturing condition,molecules such as proteins are denatured in a solution containing adetergent (SDS). In these conditions, for example, proteins are unfoldedand coated with negatively charged detergent molecules. Proteins inSDS-PAGE are then separated on the sole basis of their size. The proteinmigrates as bands based on size. Each band can be detected using stainssuch as Coomassie blue dye or silver stain. Unlike denaturing methods,native gel electrophoresis does not use a charged denaturing agent.Under native condition, molecules such as proteins maintain theirnatural structures. The molecules being separated therefore differ notonly in molecular mass and intrinsic charge, but also thecross-sectional area, and thus experience different electrophoreticforces dependent on the shape of the overall structure. For proteins,since they remain in the native state they may be visualized not only bygeneral protein staining reagents but also by specific enzyme-linkedstaining.

For purposes of the present invention, the term “gene” refers to anucleic acid (e.g., DNA or RNA) sequence that comprises coding sequencesnecessary for the production of an RNA or a polypeptide or itsprecursor. The term “portion,” when used in reference to a gene, refersto fragments of that gene. The fragments may range in size from a fewnucleotides to the entire gene sequence minus one nucleotide.

For purposes of the present invention, the term “glycosylation” refersto attachment of monosaccharides and oligosaccharides to a proteinbackbone via a glycosidic linkage. Glycosylation is a post-translationmodification. The term “hyper glycosylated” refers to more extensiveglycosylation when compared to the “normal” glycosylation observed inthe native-like cleaved trimers. These differences can be observed asdifferences in the mobility upon polyacrylamide gel electrophoresis. Inembodiments of the present invention, the term “fully glycosylated”refers to glycosylations that occur in the HEK293 cell, a humanembryonic kidney cell line used for production of gp140. “Partialglycosylation” occurs in the GnTI⁻cell line. The GnTI⁻cell line lacksthe N-acetylglucosamine transferase 1 enzyme, hence it cannot introducecomplex glycosylations.

For purposes of the present invention, the term “gp120” and the term“envelope glycoprotein gp120” refers to a glycoprotein exposed on thesurface of the HIV envelope. The gp120 in its name comes from itsmolecular weight of 120 kDa. Gp120 is essential for virus entry intocells as it plays a vital role in attachment to specific cell surfacereceptors. Gp120 is coded by the HIV env gene, which is around 2.5 kblong and codes for around 850 amino acids. The primary env product isthe protein gp160, which gets cleaved to gp120 (˜480 amino acids) andgp41 (˜345 amino acids) in the endoplasmatic reticulum by the cellularprotease furin. The crystal structure of core gp120 shows anorganization with an outer domain, an inner domain with respect to itstermini and a bridging sheet. Gp120 is anchored to the viral membrane,or envelope, via non-covalent bonds with the transmembrane glycoprotein,gp41. Three gp120s and gp41s combine in a trimer of heterodimers to formthe envelope spike, which mediates attachment to and entry into the hostcell.

For purposes of the present invention, the term “human immunodeficiencyvirus” and the term “HIV” refer to a virus of the genus Lentivirinae,part of the family of Retroviridae, and includes, but is not limited to,HIV type 1 (HIV-1) and HIV type 2 (HIV-2), two species of HIV thatinfect humans. The HIV-I virus may represent any of the known majorsubtypes or clades (e.g., Classes A, B, C, D, E, F, G, J, and H) oroutlying subtype (Group 0). Also encompassed are other HIV-I subtypes orclades that may be isolated. An “HIV isolate” refers to a type of HIVvirus that has been separated and identified from other species of HIV.

For purposes of the present invention, the term “HIV-1 gp140,” the term“gp140,” and the term “gp140 envelope protein” refer to a protein havingtwo disulfide-linked polypeptide chains, the first chain comprising theamino acid sequence of the HIV gp120 glycoprotein and the second chaincomprising the amino acid sequence of the water-soluble portion of HIVgp41 glycoprotein (“gp41 portion”). In embodiments of the presentinvention, HIV gp140 proteins include, but are not limited to, proteinswherein the gp41 portion comprises a point mutation such as I559P. I559Prefers to a mutation introduced to change Isoleucine at position 559 toProline.

For purposes of the present invention, the term “HIV-1 gp41,” the term“gp41,” the term “glycoprotein 41,” and the term “gp41 subunit” refersto a subunit of the envelope protein complex of retroviruses, includingHuman immunodeficiency virus (HIV). These terms include, but are notlimited to: 1) an entire gp41 polypeptide including the transmembraneand cytoplasmic domains; 2) a “gp41 ectodomain” (gp41ECTo); (3) a gp41modified by deletion or insertion of one or more glycosylation sites;(4) a gp41 modified so as to eliminate or mask the well-knownimmunodominant epitope; (5) a gp41 fusion protein; and (6) a gp41labeled with an affinity ligand or other detectable marker. As usedherein, “ectodomain” means the extracellular region of a transmembraneprotein exclusive of the transmembrane spanning and cytoplasmic regions.

For purposes of the present invention, the term “host cell” and the term“host” refer to 1) a cell that harbors foreign molecules, viruses, etc.;2) a cell that has been introduced with DNA or RNA, such as a bacterialcell acting as a host cell for the DNA isolated from a bacteriophage.For example, a host cell may be a living cell in which a virus such asHIV-1 reproduces.

For purposes of the present invention, the term “incorporate” refers toinsert a fragment of a first nucleic acid into a fragment of a secondnucleic acid.

For purposes of the present invention, the term “immunogen” and the term“immunogenic composition” refer to a substance or material (includingantigens) that is able to induce an immune response alone or inconjunction with an adjuvant. Both natural and synthetic substances maybe immunogens.

For purposes of the present invention, the term “immune response” refersto any response to an antigen or antigenic determinant by the immunesystem of a subject (e.g., a human). Exemplary immune responses includehumoral immune responses (e.g., production of antigen-specificantibodies, e.g., neutralizing antibodies (NAbs)) and cell-mediatedimmune responses (e.g., lymphocyte proliferation).

For purposes of the present invention, the term “junction,” the term“junction fragment,” the term “junction sequence,” and the term“junction peptide” are interchangeable and refer to a region or afragment or a portion of peptide between two subunits or sections withina polypeptide. The two subunits or sections meet or join via thejunction fragment.

For purposes of the present invention, the term “ligand” refers to anorganic molecule that donates the necessary electrons to form coordinatecovalent bonds with metallic ions. Ligand also refers to an ion, amolecule, or a molecular group that binds to another chemical entity tofor a larger complex.

For purposes of the present invention, the term “linked” refers to acovalent linkage between two polypeptides in a fusion protein. Thepolypeptides are typically joined via a peptide bond, either directly toeach other or via one or more additional amino acids.

For purposes of the present invention, the term “linker” and the term“peptide linker” are interchangeable and refer to short peptidesequences that occur between functional protein domains and link thefunctional domains together. Linkers designed by researchers aregenerally classified into three categories according to theirstructures: flexible linkers, rigid linkers, and in vivo cleavablelinkers. A flexible linker is often composed of flexible residues likeglycine and serine so that the adjacent protein domains are free to moverelative to one another. A linker also may play a role in releasing thefree functional domain in vivo (as in in vivo cleavable linkers).Linkers may offer many other advantages for the production of fusionproteins, such as improving biological activity, increasing expressionyield, and achieving desirable pharmacokinetic profiles. The compositionand length of a linker may be determined in accordance with methods wellknown in the art and may be tested for efficacy. A linker may be fromabout 3 to about 15 amino acids long. In some embodiments of the presentinvention, a linker may be about 5 to about 10 amino acids long,however, longer linker may be used in embodiments of the presentinvention.

For purposes of the present invention, a “long linker” refers to alinker that is sufficiently long that a tag that is fused to a gp140protein is accessible for binding by a binding molecule. In someembodiments of the present invention, a long linker may be more than 3amino acids in length. In some embodiments, a long linker may be 20 ormore amino acids in length. In some embodiments of the presentinvention, a long linker may be 23 or more amino acids in length. Insome embodiments, a long linker may be 27 or more amino acids in length.

For purposes of the present invention, the term “mimic” refers to have asimilar structure. For example, according to embodiment of the present,when a trimer formed by three copies of protein molecule of arecombinant HIV gp140 protein mimics native HIV Envelope protein (Env)gp140 trimer, it means that the trimer formed by three copies of proteinmolecule of a recombinant HIV gp140 protein resemble the trimericstructure formed by native HIV gp140 at natural condition. A trimerformed by recombinant HIV gp140 protein that mimics native HIVgp140trimer structure (the native Env spike) may be able to elicitneutralizing antibody responses in immunized animals.

For purposes of the present invention, the term “modified” and the term“mutant” when made in reference to a gene or to a gene product refer,respectively, to a gene or to a gene product which displaysmodifications in sequence and/or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product.

For purposes of the present invention, the term “monomer” refers to amolecule that may bind chemically to other molecules to form a polymer.The term “monomeric protein” may also be used to describe one of theproteins making up a multiprotein complex.

For purposes of the present invention, the term “mutation” refers to achange in the polypeptide sequence of a protein or in the nucleic acidsequence.

For purposes of the present invention, the term “native-like” refers toresemble a naturally existing product or a structure of the nativeproduct.

For purposes of the present invention, the term “neutralizing antibody(Nab)” refers to an antibody which either is purified from, or ispresent in, serum and which recognizes a specific antigen (e.g., HIV Envglycoprotein, such as a gp140 polypeptide or a gp120 polypeptide) andinhibits the effect(s) of the antigen in the host (e.g., a human). Asused herein, the antibody can be a single antibody or a plurality ofantibodies.

For purposes of the present invention, the term “Ni-based resin” and theterm “nickel based resin” refer to a nickel-charged resin that can beused in purification of recombinant proteins carrying a His-tag.Ni-based resins include Ni-NTA agarose or beads, and other Ni-IDAresins. Histidine residues in the His-tag bind to the vacant positionsin the coordination sphere of the immobilized nickel ions with highspecificity and affinity. For example, cleared cell lysates containing aHis-tagged recombinant protein may be loaded onto a Ni-NTA agarose.His-tagged proteins are bound, and other proteins pass through thematrix. After washing, His-tagged proteins are eluted in buffer undernative or denaturing conditions.

For purposes of the present invention, the term “nucleic acid” and theterm “polynucleotide,” as used interchangeably herein, refer to polymersof nucleotides of any length, and include DNA and RNA. The nucleic acidbases that form nucleic acid molecules can be the bases A, C, G, T andU, as well as derivatives thereof. Derivatives of these bases are wellknown in the art. The term should be understood to include, asequivalents, analogs of either DNA or RNA made from nucleotide analogs.The term as used herein also encompasses cDNA, that is complementary, orcopy, DNA produced from an RNA template, for example by the action ofreverse transcriptase. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and their analogs.

For purposes of the present invention, the term “oligomer,” when used inthe context of a protein and/or polypeptide, refers to, but is notlimited to, a protein or polypeptide having at least two subunits.Oligomers include, but are not limited to, dimers, trimers, tetramers,pentamers, hexamers, heptamers, octamers, nonamers, decamers and thelike. An oligomer can be a macromolecular complex formed by non-covalentbonding of few macromolecules like proteins or nucleic acids. In thissense, a homo-oligomer would be formed by few identical molecules and bycontrast, a hetero-oligomer would be made of three differentmacromolecules.

For purposes of the present invention, the term “operably linked,” theterm “operably associated,” and the term “functionally linked” are usedinterchangeably and refer to a functional relationship between two ormore DNA segment. Particularly, “operably linked” may refer to place afirst nucleic acid sequence in a functional relationship with the secondnucleic acid sequence. For example, a promoter/enhancer sequence,including any combination of cis-acting transcriptional control elementsis operably associated to a coding sequence if the promoter/enhancersequence affects the transcription or expression of the coding sequencein an appropriate host cell or other expression system. Promoterregulatory sequences that are operably linked to the transcribed genesequence are physically contiguous to the transcribed sequence.

For purposes of the present invention, the term “optimize” refers todetermining conditions for the maximal production of gp140 protein inthe culture medium. Optimization of gp140 protein may involvemodification of the amino acid sequence of a gp140 to determineconditions for the maximal production of modified gp140 protein in theculture medium.

For purposes of the present invention, the term “optimized polypeptide”refers to an polypeptide that is not a naturally-occurring peptide,polypeptide, or protein, such as a non-naturally occurring viralpolypeptide (e.g., a gp140 polypeptide of the invention). Optimizedviral polypeptide sequences are initially generated by modifying theamino acid sequence of one or more naturally-occurring viral geneproducts (e.g., peptides, polypeptides, and proteins, e.g., a viral Envpolypeptide, e.g., a viral Env1, Env2, and/or Env3 polypeptide). Thus,the optimized viral polypeptide may correspond to a “parent” viral genesequence; alternatively, the optimized viral polypeptide may notcorrespond to a specific “parent” viral gene sequence but may correspondto analogous sequences from various strains or quasi-species of a virus.Modifications to the viral gene sequence that can be included in anoptimized viral polypeptide include amino acid additions, substitutions,and deletions.

For purposes of the present invention, the term “polymer” refers to acompound or a mixture of compounds comprising many repeating subunits,known as monomers.

For purposes of the present invention, the term “polypeptide” and theterm “protein” are used interchangeably herein to refer to a polymer ofamino acid residues. The terms encompass amino acid polymers in whichone or more amino acid residues are artificial chemical mimetic of acorresponding naturally occurring amino acids, as well as to naturallyoccurring amino acid polymers and non-naturally occurring amino acidpolymer.

For purposes of the present invention, the term “promoter” refers to aregulatory DNA sequence generally located upstream of a gene thatmediates the initiation of transcription by directing RNA polymerase tobind to DNA and initiating RNA synthesis. A promoter may be aconstitutive promoter or an inducible promoter. A constitutive promoter(e.g. a viral promoter) is always active. For example, humancytomegalovirus (CMV) promoter drives constitutive protein expression inorganisms, such as human cells. An inducible promoter is not alwaysactive. Some inducible promoters are activated by physical means such asthe heat shock promoter.

For purposes of the present invention, the term “protomer” refers to astructural unit of an oligomeric protein. Protomer describes the factthat in oligomeric proteins some subunits are closer togetherstructurally, and work closer together functionally, than others. Forexample, an HIV Env trimer encompasses three molecules of heterodimerthat is formed by a gp120 subunit and a gp41 subunit, each dimerencompassing a gp120 and a gp41 may be called a protomer.

For purposes of the present invention, the term “protein purification”refers to a series of processes intended to isolate one or a fewproteins from a complex mixture, such as cell culture media, cells,tissues or whole organisms, etc. Usually a protein purification protocolcontains one or more chromatographic steps. The basic procedure inchromatography is to flow the solution containing the protein through acolumn packed with various materials. Different proteins interactdifferently with the column material, and can thus be separated by thetime required to pass the column, or the conditions required to elutethe protein from the column. Many purification strategies exist. Forexample, a protein can be attached with an antigen peptide tag byengineering and be purified using an antibody against the antigenpeptide tag. Usually, during purification, the protein with an antigenpeptide tag can be added on a column loaded with resin that is coatedwith an antibody or by incubating with a loose resin that is coated withan immobilizing antibody. This particular procedure is known asimmunoprecipitation. Immunoprecipitation is quite capable of generatingan extremely specific interaction which usually results in binding onlythe desired protein. The purified tagged proteins can then easily beseparated from the other proteins in solution and later eluted back intoclean solution.

For purposes of the present invention, the term “purified” refers to thecomponent in a relatively pure state, e.g. at least about 90% pure, orat least about 95% pure, or at least about 98% pure.

For purposes of the present invention, the term “recombinant vaccine”refers to a vaccine made by genetic engineering, the process and methodof manipulating the genetic material of an organism. Usually, arecombinant vaccine encompasses one or more protein antigens that haveeither been produced and purified in a heterologous expression system(e.g., bacteria or yeast) or purified from large amounts of thepathogenic organism. The vaccinated person produces antibodies to theone or more protein antigens, thus protecting him/her from disease.

For purposes of the present invention, the term “recombinant” refers toa genetic material formed by a genetic recombination process. A“recombinant protein is made through genetic engineering. A recombinantprotein is coded by a DNA sequence created artificially. A recombinantprotein is a protein that is coded by a recombinant nucleic acidsequence. A recombinant nucleic acid sequence has a sequence from two ormore sources incorporated into a single molecule.

For purposes of the present invention, the term “regulatory region”refers to a segment of a nucleic acid molecule which is capable ofincreasing or decreasing the expression of specific genes within anorganism. A regulatory sequence may include enhancer/silencer, operator,and promoter regions which regulate the transcription of the gene intoan mRNA.

For purposes of the present invention, the term “secretion” and the term“secretion of a protein” or refers to transport a protein synthesized bya cell from intracellular of the cell into the extracellular space.

For purposes of the present invention, the term “secretion signalpeptide,” the term “secretion peptide,” the term “signal peptide,” andthe term “secretion signal sequence” are used interchangeably and referto a short (about 5-30 amino acids long) peptide present at theamino-terminus (N-terminus) of secreted and membrane-bound proteins. Asecretion signal peptide present at a majority of newly synthesizedproteins that are destined towards the secretory pathway. A signalpeptide directs a newly synthesized protein to the secretory pathway.The cleavage of the signal peptide from a mature protein may occurduring or after completion of translocation to generate a free signalpeptide and a mature protein. The free signal peptides are then digestedby specific proteases. The signal peptide consists of three regions: anamino-terminal polar region (N region), where frequently positivecharged amino acid residues are observed, a central hydrophobic region(H region) of 7-8 amino acid residues and a carboxy-terminal region (Cregion) that includes the cleavage site.

For purposes of the present invention, the term “solid matrix” refers toa solid phase such as a gel matrix, often of agarose, used in affinitypurification of a protein. For example, a ligand such as a nickel (Ni)can be coupled to an agarose to form a Ni-based agarose bead. Duringaffinity purification process, molecules of interest such as aHis-tagged target protein or its oligomer can be trapped or captured ona Ni-based agarose bead and be separated from a mixture containing theHis-tagged target protein. The trapped molecules of interest can be thenreleased from the Ni-based agarose in a process known as elution.

For purposes of the present invention, the term “steric hindrance”refers to the prevention or retardation of inter molecule interactionsas a result of the spatial structure of a molecule. Steric hindranceoccurs when the 3-dimensional (3-D) shape of a molecule in large sizeprevents ready access to the molecule in large size by a molecule insmaller size. For example, a peptide fragment such as a protein tagwithin a large protein molecule may be sterically hindered to bind to abinding molecule because the 3-D conformation or shape of the largeprotein molecule blocks the access of the binding molecule to thepeptide fragment.

For purposes of the present invention, the term “stimulate,” the term“immuno-stimulate” refers to induce the activation or increase theactivity of any components in an immune system. For example, T cellactivation requires at least two signals to become fully activated. Thefirst occurs after engagement of the T cell antigen-specific receptor(TCR) by the antigen-major histocompatibility complex (MHC), and thesecond by subsequent engagement of co-stimulatory molecules. Oncestimulated, the T cells will recognize the antigen or vaccine usedduring stimulation or activation of the T cells.

For purposes of the present invention, the term “STREP-TAG® II” refersto an eight-residue minimal peptide sequence(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 2)) that exhibits intrinsicaffinity toward streptavidin and can be fused to recombinant proteins invarious fashions.

For purposes of the present invention, the term “subunit” refers to aseparate polypeptide chain that makes a certain protein which is made upof two or more polypeptide chains joined together. In a protein moleculecomposed of more than one subunit, each subunit can form a stable foldedstructure by itself. The amino acid sequences of subunits of a proteincan be identical, similar, or completely different.

For purposes of the present invention, the term “tag,” the term “peptidetag,” and the term “protein tag” refer to, but are not limited to, apolypeptide sequence that can be added to another polypeptide sequencefor a variety of purposes. In certain exemplary embodiments, a proteintag may be removed from a larger polypeptide sequence when it is nolonger needed. Protein tags include, but are not limited to, affinitytags, epitope tags, etc. Affinity tags are appended to proteins so thatthey can be purified from their crude biological source using anaffinity technique. For example, an His tag is a widely used proteintag. An His tag has a DNA sequence specifying a string of six to ninehistidine residues (SEQ ID NO: 16) and is frequently used in vectors forproduction of recombinant protein. The result is expression of arecombinant protein with an His tag such as a 6×His tag (SEQ ID NO: 4)fused to N- or C-terminus of the recombinant protein. ExpressedHis-tagged proteins can be purified and detected easily because thestring of histidine residues binds to several types of immobilized metalions, including nickel, cobalt and copper, under specific bufferconditions. In addition, anti-His-tag antibodies are commerciallyavailable for use in assay methods involving His-tagged proteins. Ineither case, the tag provides a means of specifically purifying ordetecting the recombinant protein without a protein-specific antibody orprobe. Other affinity tags include, but are not limited to, chitinbinding protein (CBP), maltose binding protein (MBP),glutathione-s-transferase (GST) and the like. Epitope tags are shortpeptide sequences which are chosen because high-affinity antibodies canbe reliably produced in many different species. Epitope tags may be usedin antibody purification. In some situations, after purification, tagsare commonly removed by approaches such as specific proteolysis, etc.

For purposes of the present invention, the term “tagged protein” refersto a recombinant protein that is fused with a tag.

For purposes of the present invention, the term “target” refers to aliving organism or a biological molecule to which some other entity,such as a small molecule like a ligand or an antibody, is directedand/or binds.

For purposes of the present invention, the term “transfection” refers toa variety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell by calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,lipofection (e.g., using commercially available reagents such as, forexample, LIPOFECTIN® (Invitrogen Corp., San Diego, Calif.),LIPOFECTAMINE® (Invitrogen), FUGENE® (Roche Applied Science, Basel,Switzerland), JETPEI™ (Polyplus-transfection Inc., New York, N.Y.),EFFECTENE® (Qiagen, Valencia, Calif.), DREAMFECT™ (OZ Biosciences,France) and the like), electroporation (e.g., in vivo electroporation),etc. Suitable methods for transfecting host cells can be found inSambrook, et al., (“Molecular Cloning: A Laboratory Manual.” 2nd, ed.,Cold Spring harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989), and other laboratory manuals.

For purposes of the present invention, the term “trimer” refers to “aprotein trimer.” A protein trimer is a macromolecular complex formed bythree macromolecules like proteins. A homo-trimer would be formed bythree identical molecules. A hetero-trimer is formed by three differentmacromolecules. For example, a native and functional HIV-1 envelopeglycoprotein (Env) complex is present on the virus surface as a trimer,or trimeric HIV-1 envelope glycoproteins. The trimeric HIV-1 envelopeglycoproteins (Env) that are displayed on human and simianimmunodeficiency viruses (HIV and SIV, respectively) are heterodimers ofthe transmembrane glycoprotein (gp41) and a surface glycoprotein(gp120). The glycoproteins gp120 and gp41 are synthesized initially as asingle gp160 polypeptide that is subsequently cleaved to generate thenoncovalently associated gp120/gp41 complex.

For purposes of the present invention, the term “trimerization” refersto a process of polymerization resulting in a trimer.

For purposes of the present invention, the term “truncation” refers toelimination of the N- or C-terminal portion of a protein by proteolysisor manipulation of the structural gene, or premature termination ofprotein elongation due to the presence of a termination codon in itsstructural gene as a result of a nonsense mutation.

For purposes of the present invention, the term “uncleaved” refers torefers to a protein or polypeptide that is not cleaved by furin. Forexample, the furin cleavage site REKR (SEQ ID NO: 14) between a gp120and gp41 may be mutated to SEKS (SEQ ID NO: 15), which results in agp140 that is not cleaved into gp120 and gp41 by furin.

For purposes of the present invention, the term “vaccine” refers to abiological compound or an agent used to improve immunity to a particulardisease. The agent injected into a human or animal body stimulates thebody's immune system to recognize the agent as foreign, destroy it, andkeep a record of it, so that the immune system can more easily recognizeand destroy any of these microorganisms that it later encounters. Forexample, an HIV vaccine improves the production of neutralizing anti-HIVantisera.

For purposes of the present invention, the term “vector” and the term“suitable vector” refer to any vehicle used to transfer genetic materialto a target cell, such as a plasmid or a viral vector. A vector may alsobe cloning vector or expression vector. A vector may incorporate anucleic acid sequence encoding a polypeptide or protein and any desiredcontrol sequences. It may bring about the expression of the nucleic acidsequence. The choice of the vector will typically depend on thecompatibility of the vector with a host cell into which the vector is tobe introduced. A vector may include, but is not limited to, a virus(e.g., adenovirus or poxvirus), naked DNA, oligonucleotide, cationiclipid (e.g., liposome), cationic polymer (e.g., polysome), virosome,nanoparticle, or dentrimer. The nucleic acid material of a viral vectormay be encapsulated, e.g., in a lipid membrane or by structural proteins(e.g., capsid proteins), that may include one or more viral polypeptides(e.g., an envelope glycoprotein). The viral vector can be used to infectcells of a subject, which, in turn, promotes the translation of theheterologous gene(s) of the viral vector into a protein product (e.g.,one or more of the recombinant gp140 Env polypeptides described herein,such that a stabilized trimer of the invention is formed).

For purposes of the present invention, the term “virus” refers to aninfectious agent that is unable to grow or reproduce outside a host celland that infects mammals (e.g., humans) or birds.

For purposes of the present invention, the term “wild-type” and the term“native,” when made in reference to a gene, refers to a gene that hasthe characteristics of a gene isolated from a naturally occurringsource. The term “wild-type” and the term “native,” when made inreference to a gene product, refers to a gene product that has thecharacteristics of a gene product isolated from a naturally occurringsource. The term “naturally-occurring” as applied to an object refers tothe fact that an object can be found in nature. A wild-type gene isfrequently that gene which is most frequently observed in a populationand is thus arbitrarily designated the “normal” or “wild-type” form ofthe gene.

DESCRIPTION

Embodiments disclosed herein provide an approach to capture variousforms of recombinant HIV-1 envelope protein (gp140) and recombinantgp140 trimers. A recombinant HIV-1 gp140 can be engineered based on anHIV-1 envelope sequence from any different clades or strains of HIV-1.In some embodiments, engineered HIV-1 envelope protein (gp140) fromdifferent clades or strains of HIV-1 are purified from a culture mediumunder mild conditions that cause minimal, if any, perturbation to thestructure of oligomeric state of gp140.

AIDS (acquired immune deficiency syndrome) caused by the humanimmunodeficiency virus-1 (HIV-1) is a global epidemic. More than 30million people worldwide currently live with HIV infection and nearly 2million people die of AIDS every year. Nine genetic subtypes andnumerous circulating recombinant forms have been identified. Coupledwith this diversity is the extraordinary evolution of the viral envelopeprotein (Env) in response to host immune pressures. Designing an Envimmunogen that can stimulate antibodies (Abs), which in turn can blockentry of genetically diverse HIV-1 viruses, has remained as the “holygrail” of the HIV vaccine field.^(1,2)

The trimeric Env spike of the HIV-1 virion is the virus entry machine. Atrimeric Env spike of the HIV-1 virion is a trimer of hetero-dimercomposed of glycoproteins gp120 and gp41 produced by cleavage of theprecursor protein gp160.^(3,4) The gp41 is a transmembrane glycoproteindisplayed on the surface of the viral membrane. The entry of HIV-1involves a series of well-orchestrated interactions between theseproteins and the receptor molecules present on the target cell.⁵ Thefirst step might be the capture of the virus through interactionsbetween the V1V2 domain of gp120 and a surface molecule such as the α4β7integrin of the mucosal T lymphocytes.^(6,7) This first step might bringthe virus into close proximity to CD4, the primary receptor for HIV-1.Binding to CD4 causes a conformational change in gp120, exposing a sitein the V3 domain that binds to the chemokine co-receptor CCR5 orCXCR4.⁸⁻¹³ A series of conformational changes ensue resulting in theinsertion of the gp41 fusion peptide into the host cell membrane.¹⁴ Theviral lipid bilayer fuses with the plasma membrane releasing thenucleocapsid core into the target cell.¹⁵ Therefore, Env-specific Absthat can interfere with any of the steps common to diverse HIV-1 virusescan prevent transmission of HIV into the host.

Several human monoclonal Abs (mAbs), referred to as broadly neutralizingAbs (BnAbs), have been discovered that can neutralize infection of alarge spectrum of genetically diverse HIV-1 viruses. These include, forinstance, BnAbs b12 and VRC01 that bind to the CD4 binding site (bs) ofgp120, 2F5 and 4E10 that bind to the membrane proximal external region(MPER) of gp41, and PG9 and PG16 that bind to the V1V2 domains of thetrimer.^(16,17,18,19) Most of these Abs recognize conformationalepitopes and are produced either by “elite controller” individuals withchronic HIV infections, or by selection of rare B cell clones present inHIV-1 infected individuals.²⁰ They also exhibit unusual features such asthe presence of a long heavy chain 3 complementarity determining region(CDR) covering a large area of the epitope as well as dozens of somaticmutations introduced by a process known as “affinity maturation” drivenby the evolving envelope protein.²¹ Attempts to induce such BnAbs inanimal models, or in humans, by vaccination with recombinant Envimmunogens have thus far failed.^(22,23,24,25)

One reason for this failure may be that the subunit Env immunogens donot recapitulate the trimeric structure of the native Env spike presenton the HIV-1 virion.²⁶ It has been hypothesized that exposure to“native” trimers can lead to activation and expansion of rare B cellclones of the right BnAb lineage.²⁷ Furthermore, such a trimer can alsobe used as a scaffold to engineer variants that represent a commonstructure present in diverse HIV-1 strains. However, production of Envtrimers that mimic the native spike has remained a challenge, in partbecause the recombinant trimers either are unstable or aggregate.

Recently, Ringe et al discovered that an HIV-1 subtype A isolate BG505naturally produces relatively stable trimers.²⁶ By further stabilizingthe trimer with mutations that crosslink cleaved gp120 and gp41 througha disulfide bond (SOSIP mutations), “native-like” trimers areproduced.²⁶ These “native-like” trimers are then captured by the BnAb2G12 and purified.^(26,28) The structures of the trimers complexed withvarious BnAbs are determined by cryo-electron microscopy (EM) and X-raycrystallography.^(29,30) However, this Ab-based approach has inherentlimitations since the gp140 structure and it epitope signatures varyfrom one HIV strain to another because of the amino acid sequencedifferences (HIV envelope protein is frequently mutated). Thus, thisAb-based approach is not as effective with diverse HIV-1 strains thatmight differ in the epitope signature.³¹ For example, to create theepitope binding site for 2G12, the wild-type BG505 gp140 is mutated bychanging Thr332 to Asn.²⁶ Moreover, the BnAb 2G12 is not readilyavailable, prohibitively expensive, and not practical for vaccinemanufacture.

Based on investigations on the design of HIV-1 Env immunogens andefficient vaccine delivery systems,^(32,33,34) embodiments of thepresent invention provide a new system to isolate and characterize Envtrimers, potentially from any HIV-1 virus strain. First, an Envrecombinant protein is constructed by attaching a highly specific tagsuch as an eight amino acid (aa) STREP-TAG® II separated from thecarboxy terminus (C-terminus) of a recombinant gp140 by a long linker.The long linker makes the highly specific tag accessible for binding bya binding molecule bound on a solid matrix. The Env recombinant proteincan be efficiently captured by a bind molecule such as STREP-TACTIN®, aspecially-engineered streptavidin ligand, directly from a culture medium(culture supernatant). The Env recombinant protein bound toSTREP-TACTIN® can then be dissociated under mild conditions to generate˜95% pure Env recombinant protein in a single step. Second, embodimentsof the present invention develop a screening strategy to optimize anyEnv recombinant protein construction for maximal trimer production. Forexample, the JRFL Env gp140 selected by this approach produce ˜70% ofrecombinant gp140 as trimers. Third, the cleaved JRFL Env recombinanttrimers exhibit the classic three-blade propeller shape³⁵ and theirbiochemical and antigenic properties are consistent with the nativetrimers. Fourth, according to embodiments, both cleavage and properglycosylation are critical for maturation of recombinant gp140 intoauthentic trimers. Although recombinant gp140 can trimerize withoutcleavage, uncleaved trimers enter aberrant pathways generatinghyper-glycosylated and conformationally heterogeneous particles.Finally, the trimers, including the cleaved propeller trimers, showmicro-heterogeneity in the extent of gp41 glycosylation.

Tags such as hexa-histidine (SEQ ID NO: 4) or STREP-TAG® II (SEQ ID NO:2) have often been fused to a protein of interest. A binding moleculethat is specific to the tag and bound to a solid matrix such as anagarose bead can be used to capture the tag and the protein attached toit. However, previous attempt to selectively capture and purify HIV-1gp140 envelope protein, by using either a hexa-histidine tag (SEQ ID NO:4) or a STREP-TAG® fused to HIV-1 gp140, from a crude preparation IIfails. The reason for the failure may be that a tag such as STREP-TAG®II or hexa-histidine (SEQ ID NO: 4) is occluded when it is attached tothe base of the gp140 structure. Consequently, the large STREP-TACTIN®beads used to capture the STREP-TAG® II (or the Nickel beads used tocapture the histidine tag) may clash with the carboxy terminus of gp140to which the tag is attached. The glycan shield containing up to 12large complex glycans that are also attached to the carboxy terminalhelix may make the tag even more sterically hindered, which makes theclashes even worse.

According to some embodiments of the present invention, separating a tagfrom the base of a gp140 envelope trimer by a long linker solves theproblem that a tag is sterically hindered by the base of gp140. FIG. 1is a schematic diagram showing recombinant trimer 110 of envelope gp140protein fused to tag 112 through long linker 116 according to oneembodiment of the present invention in comparison with a recombinanttrimer 110 of envelope gp140 protein fused to tag 112 without longlinker 116. As shown in FIG. 1, tags 112 not separated from the base ofrecombinant trimer 110 are sterically hindered by the base ofrecombinant trimer 110 of envelope gp140 protein and cannot be capturedby binding molecule 122 bound on solid matrix 124. Contrarily, by beinglocated further away from the base of recombinant trimer 110 of envelopegp140 protein, tag 112 can be captured by binding molecule 122 bound onsolid matrix 124. Binding molecule 122 specifically binds to tag 112 andtherefore catches recombinant trimer 110 of envelope gp140 protein whichis fused to tag 112 through long linker 116. The separation of tag 112from the base of recombinant trimer 110 of envelope gp140 proteinthrough long linker 116 allows the capture of near homogenousrecombinant trimers 110 directly from a culture medium under a mildcondition. Recombinant trimer 110 of envelope gp140 protein purifiedfrom the culture medium mimics a native HIV-1 envelope trimer. Tocapture recombinant trimer 110 of envelope gp140 protein, bindingmolecule 122 may be a molecule that specifically interacts with tag 112,such as a molecule of streptavidin, nickel, etc. Solid matrix 124 may bean agarose bead. In some embodiment of the present invention, beads maybe contained in a chromatography column as a stationary phase to purifyrecombinant trimers.

FIG. 1 is only an illustrative example of a representative recombinanttrimer that can be purified directly from a culture medium under mildcondition. A tag that can be used to fuse with a recombinant HIV-1 gp140for purification is not limited to STREP-TAG® II, which is shown in FIG.1 as an example. Any tag that can be captured by a binding moleculeimmobilized on a solid matrix can be used. For example, in addition toSTREP-TAG® II, a tag may be an affinity tag such as a strep-tag otherthan STREP-TAG® II, a hexa-histidine tag (SEQ ID NO: 4), anocta-histidine tag (SEQ ID NO: 3), a chitin binding protein (CBP) tag, amaltose binding protein (MBP) tag, a polyglutamate tag, aglutathione-S-transferase (GST) tag, a FLAG-tag, an SBP-tag, a softag,etc. A tag may also be an epitope tag such as a V5-tag, Myc-tag, HA-tag,E-tag, VSV-tag, etc. The tags listed herein are not exclusive. One ofordinary skill in the art would readily appreciate that any tag that issuitable for purification of a recombinant trimer described herein maybe utilized.

Particularly, in some embodiments, a recombinant protein comprises arecombinant HIV-1 gp140, a linker, and a tag, wherein the recombinantHIV-1 gp140 is fused to the tag through the linker between the tag andC-terminus of the recombinant HIV-1 gp140. The linker is long enough tomake the tag accessible for finding by a binding molecule bound on asolid matrix; or in other words, the linker is sufficiently long so thatthe tag is accessible for binding by a binding molecule bound on a solidmatrix. The length of the linker varies according to the needs ofpurifying different types of trimers. The linker may be a flexiblelinker or a rigid linker. Embodiments provide various linkers withdifferent length. In some embodiments, the linker may be 20 or moreamino acids in length. In some embodiments, the linker is 23 or moreamino acids in length. In some embodiments, the linker. In someembodiments, the linker may be 27 or more amino acids in length. Forexample, Foldon sequence (SEQ ID NO: 1) which has 27 amino acids canalso be used as a linker.

The recombinant proteins can be expressed in cells and be secreted fromthe cells into culture medium in which the cells grow. To secret therecombinant protein from cells expressing the recombinant protein intocell culture medium in which the cells grow, a secretion signal peptidemay be attached to N-terminus of the recombinant HIV-1 gp140. In someembodiments, the secretion signal peptide is human CD5 secretion signalpeptide. Once expressed in cells, the recombinant protein is secretedinto the culture medium in which the cells expressing the recombinantprotein grow. The recombinant protein then assembles into recombinanttrimers that mimic native HIV-1 envelope trimers.

The recombinant proteins and recombinant trimers assembled in culturemedium are easy to be captured and purified with a binding moleculebound and immobilized on a solid matrix. The binding molecule thatspecifically targets to a tag separated from a recombinant gp140 proteinor a recombinant trimer base by a linker will access the tag withoutsteric hindrance. Therefore, the binding molecule catches therecombinant gp140 fused to the tag. The binding molecule that isspecific to a tag fused to a recombinant HIV-1 gp140 may beSTREP-TACTIN® or Nickel beads. The solid matrix that the bindingmolecule bound on may be agarose beads. This approach allows the captureof near-homogeneous recombinant gp140 and trimers directly from culturemedium under a mild condition. Under mild condition, the recombinantHIV-1 envelope trimer purified from the culture medium mimics a nativeHIV-1 envelope trimer.

FIG. 2 is a schematic image of gp140 expression cassette encompassingvarious exemplary linkers and tags attached to C-terminus of arecombinant HIV-1 gp140. In Panel A of FIG. 2, P_(CMV) refers to CMVpromoter and CD5 refers to secretion peptide; C1-05 are conserveddomains in gp120 subunit 210, and V1-V5 are variable domains in gp120subunit 210; Heptad repeats 1 and 2 (HR1 and HR2) and membrane proximalexternal region (MPER) are regions in gp41 subunit 220. Both gp120 andgp41 have many N-linked glycosylation sites (see trees 230 and trees240). The positions of a Furin Cleavage site is located within ajunction region 250 between the gp120 subunit 210 and the gp41 subunit220. Panel A of FIG. 2 also shows disulfide bond mutations (SOS) andI559P mutation in the gp140 expression cassette. A secretion peptide260, such as a human CD5, is fused to C1 domain at N-terminal region ofthe gp120 subunit 210. Panel B of FIG. 2 is a schematic drawingillustrates various exemplary linkers and tags attached to HIV-1 gp140C-terminus. The amino acid sequence of each tag or linker is shown inthe boxes. HRV-3C refers to human rhinovirus protease cleavage site.

FIG. 2 is only an illustrative representation of a gp140 expressioncassette encompassing a recombinant HIV-1 gp140. Tags and linkers thatcan be used for fusing with a recombinant HIV are not limited to thelinker and tag shown in FIG. 2. One of ordinary skill in the art wouldreadily appreciate that any linker and tag that are suitable forconstructing a recombinant trimer described herein may be utilized.

Embodiments provide various linkers and tags as examples. For example,tags may be a STREP-TAG® II (SEQ ID NO: 2) or a poly-His tag such as anocta-histidine (His) tag (SEQ ID NO: 3), a hexa-histidine tag (SEQ IDNO: 4), etc. In some embodiments, an octa-histidine (SEQ ID NO: 3) tagis used instead of a hexa-histidine tag (SEQ ID NO: 4) because theformer is more specific than the latter. In some embodiments, forexample as shown in FIG. 2, a linker is an (Ala)₃ linker which comprisesthree alanines (Ala) at the N-terminus of the linker sequence. Thelength of a linker to separate a tag from a recombinant gp140 variesaccording to the needs for purifying different types of recombinantgp140. In one embodiment, the sequence of the linker comprises SEQ IDNO: 5, which has 23 amino acids in length. In some embodiments, a linkermay have a sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQID NO: 9, in which the linker comprising SEQ ID NO: 8 is an HRV-3Cflexible linker, and the linker comprising SEQ ID NO: 9 is an HRV-3crigid linker. In some embodiment, a linker may be a foldon sequence(27aa) that has an amino acid sequence comprises SEQ ID. NO: 1.

As illustrated in Panel B of FIG. 2, a linker and a tag may be shown asan extended tag that encompasses both the sequence of a linker and thesequence of a tag. For example, as extended tag may be Twin STREP-TAG®(TSt), HRV-3C Twin STREP-TAG® (TSt-3C), HRV-3C Twin STREP-TAG® (TSt-3C),Long linker Twin STREP-TAG® (TSt-LL), HRV-3C Flexible linker octa-Histag (“octa-His tag” disclosed as SEQ ID NO: 3) (OHt-FL 3C) and HRV-3CRigid linker octa-His tag (“octa-His tag” disclosed as SEQ ID NO: 3)(OHt-RL 3C). Some of the linkers, for example, HRV-3C Twin STREP-TAG®(TSt-3C) and HRV-3C Twin STREP-TAG® (TSt-3C), comprise a portion of asequence from a tag. This portion of sequence functions as a part oflinker to extend the amino acid length between a tag and C-terminus of arecombinant HIV-1 gp140.

Accordingly, a series of recombinant proteins are engineered by fusingthe gp140 C-terminus to a tag, such as a hexa-histidine tag (SEQ ID NO:4), an octa-histidine tag (SEQ ID NO: 3), or a STREP-TAG® II tag, withvarious linkers in the middle to separate the tag from the gp140 base.The recombinant HIV-1 gp140 may be derived from any HIV-1 gp140. Forexample, in some embodiments, the HIV-1 clade B JRFL gp140 and clade ABG505 gp140 sequences are used as templates to engineer the recombinantproteins, respectively. Further, in some embodiments, a recombinantHIV-1 gp140 protein comprising a gp120 and a gp41 ectodomain may have asequence truncated at amino acid residue 664 (aa664) (based on HXB2numbering) or at a position not beyond aa664. The gp120 and the gp41ectodomain are joined via a junction sequence that contains a furincleavage site REKR (SEQ ID NO: 14). In some embodiments, the furincleavage site REKR (SEQ ID NO: 14) is mutated to SEKS (SEQ ID NO: 15),and such mutation results in a recombinant gp140 that is furin cleavageresistant (CR). In come embodiments, the recombinant gp140 is furincleavage proficient (CP), in which the furin cleavage site REKR (SEQ IDNO: 14) is mutated to RRRRRR (SEQ ID NO: 13).

According to some embodiments, a recombinant HIV-1 gp140 may furthercomprise three “SOSIP” mutations that include A501C, T605C, and I559P.In some embodiments, a recombinant HIV-1 gp140 may further comprise five“stabilizing” mutations which include I535M, Q543L, S553N, K567Q, andR588G. The A501C and T605C mutations create an intra-protomer disulfidebond between gp120 and gp41. The I559P mutation is a point mutationwherein the isoleucine residue at position aa559 of a polypeptide chainof an HIV-1 gp140 is replaced by a proline residue. The I559P mutationstrengthens inter-subunit interactions.

Embodiments of the present invention also provide methods to producerecombinant trimers that mimic native HIV-1 Env trimers. The recombinanttrimers can be purified from a culture medium for growing cells thatexpress and secrete the recombinant proteins described above.

In some embodiment, recombinant trimers are uncleaved recombinanttrimers. Particularly, the uncleaved recombinant trimers are assembledby a recombinant protein that is not cleaved by furin. This uncleavedrecombinant protein comprises a recombinant HIV-1 gp140 fused to a tagthrough a linker. The linker is sufficiently long so that the tag isaccessible for binding by a binding molecule bound on a solid matrix.The recombinant HIV-1 gp140 comprises two subunits: a gp120 and a gp41ectodomain that are connected together via a junction sequence (alsocalled a junction peptide). The gp41 ectodomain may further have atruncation at aa664 or have a truncation at a position not beyond aa664based on HXB2 numbering. The junction sequence between the gp120 andgp41 ectodomain is absent of a furin cleavage site REKR (SEQ ID NO: 14)but instead contains a furin cleavage resistant sequence SEKS (SEQ IDNO: 15). Therefore, the recombinant HIV-1 gp140 are furin cleavageresistant and will not be cleaved by furin. To secret the recombinantprotein into a culture medium, a secretion signal peptide such as ahuman CD5 secretion signal peptide is fused at N-terminus of therecombinant HIV-1 gp140. When the recombinant protein comprising arecombinant HIV-1 gp140 fused to a tag through a linker disclosed hereinis expressed in a cell growing in a culture medium, the recombinantprotein is secreted from the cell into the culture medium and assemblesinto trimers in the culture medium.

Embodiments also provide cleaved trimers, which are recombinant trimersof heterodimers composed of a cleaved gp120 and a cleaved gp4lectodomain. A tag is fused to C-termimus of the cleaved g41 ectodomainthrough a linker. The cleaved gp120 and cleaved gp41 ectodomain areproduced by a cleavage of a recombinant protein comprising a recombinantHIV-1 gp140 fused to a tag through a linker. The recombinant HIV-1 gp140has a gp120 and a gp41 ectodomain connected by a junction sequencebetween the gp120 and the gp41 ectodomain. The junction sequenceincludes a furin cleavage site REKR (SEQ ID NO: 14). To improve cleavageefficiency, the furin cleavage site REKR (SEQ ID NO: 14) is mutated toRRRRRR (SEQ ID NO: 13). The linker is long enough to make the tagaccessible for finding by a binding molecule bound on a solid matrix; orin other words, the linker is sufficiently long so that the tag isaccessible for binding by a binding molecule bound on a solid matrix. Insome embodiments, the linker has an amino acid sequence comprising 20amino acids in length or longer. In some embodiments, the linker has anamino acid sequence comprising 23 amino acids in length or longer. Asecretion signal peptide such as a human CD5 secretion signal peptide isfused at N-terminus of the recombinant HIV-1 gp140 fused to a tag forsecretion of the recombinant protein into culture medium one therecombinant protein is expressed in cells growing in the culture medium.As a result, when the recombinant protein is expressed in a cell, therecombinant protein is secreted from the cell into a culture medium inwhich the cell grows and is cleaved by furin into a cleaved gp120 and acleaved gp4lectodomain. The cleaved gp120 and the cleaved gp41ectodomain form a heterodimer in which the cleaved gp120 and the cleavedgp41 ectodomain are covalently associated through a disulfide bond. Theheterodimer assembles into trimers that mimic native HIV-1 envelopetrimers in the culture medium. In the heterodimer, the tag is fused tothe C-terminus of the cleaved gp41 ectodomain through the linker. Thelinker is long enough to separate the tag from the trimer base of theheterodimer, as a result, the tag is accessible for binding by a bindingmolecule bound on a solid matrix.

According to embodiments, in both cleaved and uncleaved trimers, a tagmay be a STREP-TAG® II tag, a hexa-histidine tag (SEQ ID NO: 4), or anocta-histidine tag (SEQ ID NO: 3). A linker is in the middle of the tagand the recombinant HIV-1 gp140 and separates the tag from therecombinant gp140 base. The linker is long enough to make the tagaccessible for finding by a binding molecule bound on a solid matrix; orin other words, the linker is sufficiently long so that the tag isaccessible for binding by a binding molecule bound on a solid matrix.The linker may be flexible or rigid, and varies in length according tothe needs of purifying different types of trimers. In some embodiments,a linker is 20 amino acids in length or longer. In some embodiments, alinker is 23 amino acids in length or longer. In one embodiment, alinker comprises a foldon sequence that is 27 amino acids in length (SEQID NO: 1).

In producing both cleaved and uncleaved trimers, three “SOSIP” mutationsmay be introduced into a sequence of an HIV-1 gp140 to construct arecombinant HIV-1 gp140. The “SOSIP” mutations include A501C, T605C, andI559P. In some embodiments, the recombinant HIV-1 gp140 may alsoencompass five “stabilizing” mutations (in comparing with a native HIV-1gp140). The five “stabilizing” mutations include I535M, Q543L, S553N,K567Q, and R588G. In some embodiments, the recombinant HIV-1 gp140 isengineered based on the sequence of an HIV-1 clade B gp140 such as JRFLgp140 or SF162 gp140. In some embodiment, the recombinant HIV-1 gp140 isengineered based on the sequence of an HIV-1 clade A gp140 such as BG505gp140. For example, embodiments provide a recombinant protein JRFLSOSIP(1-5).R6.664 gp140 that is a Strep-Tagged gp140 protein engineeredby using an HIV-1 clade B JRFL gp140 as a template. The recombinantprotein JRFL SOSIP(1-5).R6.664 gp140 has an amino acid sequencecorresponding to SEQ ID NO: 10. Embodiments also provide a recombinantprotein BG505 SOSIP.R6.664 gp140 which is a Strep-Tagged gp140 proteinengineered by using an HIV-1 clade A BG505 gp140 as a template. Therecombinant protein BG505 SOSIP.R6.664 gp140 has an amino acid sequencecorresponding to SEQ ID NO: 11. Embodiments also provide a recombinantprotein SF162 SOSIP.R6.664 gp140 is a Strep-Tagged gp140 proteinengineered by using an HIV-1 clade B SF162 gp140 as a template. Therecombinant protein SF162 SOSIP.R6.664 gp140 has an amino acid sequencecorresponding to SEQ ID NO: 12.

In some embodiments, a recombinant protein comprising a recombinantHIV-1 gp140 attached to a tag through a linker at C-terminus of therecombinant HIV-1 gp140 is expressed in mammalian cell lines such asHEK293F (293F), 293T, 293EXPI, CHO, HEK293S GnTI⁻ (GnTI⁻), etc. When therecombinant HIV-1 gp140 is expressed in cells growing in a culturemedium, the secretion signal peptide fused at the N-terminus of therecombinant HIV-1 gp140 helps the recombinant protein translocate frominside the cells into the culture medium in which the recombinantprotein assembles into trimers.

According to embodiments, cells expressing a recombinant protein aretransfected with a recombinant DNA encoding a recombinant proteincomprising recombinant HIV-1 gp140 fused to a tag through a linker. Thelinker is sufficiently long so that the tag is accessible for binding bya binding molecule bound on a solid matrix. The linker may be flexibleor rigid, and varies in length according to the needs of purifyingdifferent trimers. In some embodiments, the linker is at least 20 aminoacids. In some embodiments, the linker is 23 amino acids in length orlonger. In one embodiment, 293F cells are transfected with a recombinantDNA encoding a recombinant protein comprising recombinant HIV-1 gp140fused to a tag through a linker. The recombinant protein may beexpressed in the transfected 293F cells with a control of a promoter.The promoter may be a constitutive promoter or an inducible promoter. Inone embodiment, a promoter regulating the expression of the recombinantprotein is promoter CMV. Once expressed, the recombinant protein issecreted from the 293F cells into the culture medium and assembles intotrimers in the culture medium. The recombinant protein without furincleavage site will assembles into uncleaved trimers in the culturemedium. The recombinant protein with a furin cleavage site, including anenhanced furin cleavage site, will be cleaved by furin into a cleavedgp120 and a cleaved gp41 ectodomain and form a heterodimer, wherein thecleaved gp120 and the cleaved gp41 are covalently associated through adisulfide bond.

In one embodiment, GnTI⁻ cells are transfected with a recombinant DNAencoding a recombinant protein comprising recombinant HIV-1 gp140 fusedto a tag through a linker. The recombinant protein may be expressed inthe transfected GnTI⁻ cells with a control of a promoter. The promotermay be a constitutive promoter or an inducible promoter. In oneembodiment, a promoter regulating the expression of the recombinantprotein is promoter CMV. Once expressed or produced, the recombinantprotein is secreted from the GnTI⁻ cells into the culture medium. Therecombinant protein without furin cleavage site will assembles intouncleaved trimers in the culture medium. The recombinant protein with afurin cleavage site, including an enhanced furin cleavage site, will becleaved by furin into a cleaved gp120 and a cleaved gp41 ectodomain andform a heterodimer, wherein the cleaved gp120 and the cleaved gp41 arecovalently associated through a disulfide bond.

In some embodiments, a recombinant DNA encoding a recombinant HIV-1gp140 that has a furin cleavage site, including an enhanced furincleavage site RRRRRR (SEQ ID NO: 13), is co-transfected into cells witha recombinant DNA encoding a protein furin. Such co-transfectionenhances the cleavage efficiency and increases the production ofheterodimers formed by cleaved gp120 and gp41 ectodomain.

According to embodiments, a recombinant HIV-1 gp140 expressed in GnTi⁻cells is partially glycosylated, while a recombinant HIV-1 gp140expressed in 293F cells is hyper-glycosylated or fully glycosylated. Insome embodiments, the recombinant gp140 purified by the method disclosedherein is about 95% pure. According to embodiments, cleavage of gp140 isnot essential for trimerization, but it triggers a conformational changethat channels trimers into correct glycosylation pathways generatingcompact three-blade propeller-shaped trimers. Most uncleaved trimersenter aberrant pathways resulting in hyper-glycosylation andconformational heterogeneity. Embodiments of the present inventionestablish a broadly applicable system for production andcharacterization of HIV-1 trimers and generate new insights into theassembly and maturation of HIV-1 trimers that will have implications tothe design of an effective HIV vaccine. A vaccine developed from therecombinant trimer disclosed herein that mimics native spike may elicitentry-blocking antibodies and prevent HIV infection.

Corresponding to recombinant proteins described above, embodiments ofthe present invention also provide a recombinant DNA encoding arecombinant protein for making recombinant trimers that mimic nativeHIV-1 envelope trimers. Accordingly, a recombinant DNA is constructed toencompass a nucleic acid sequence encoding a recombinant protein,wherein the recombinant protein encompasses a recombinant HIV-1 gp140fused to a tag through a peptide linker at C-terminus of the recombinantHIV-1 gp140. The peptide linker is sufficiently long so that the tag isaccessible for binding by a binding molecule bound on a solid matrix. Insome embodiments, the peptide linker has a length of 20 amino acids orlonger. In some embodiments, the peptide linker has a length of 23 aminoacids or longer. A nucleic acid sequence encoding a secretion signalpeptide is attached at 5′ end of the sequence encoding the recombinantHIV-1 gp140. The recombinant DNA may be transfected into cells forexpression of the fusion protein. Cells carrying the recombinant DNAafter transfection may grow in a medium to allow the expression of therecombinant protein. The recombinant protein expressed in the cells isthen secreted from the cells into culture medium and assembles intorecombinant trimers that mimic native HIV-1 Env trimers.

According to embodiments of the present invention, the peptide linkermay vary in length according to the needs of purifying different typesof trimers. The peptide linker is selected for separating the tag fromthe recombinant HIV-1 gp140, so that a binding molecule specificallytarget the tag will not be sterically hindered to access the tag. Thepeptide linker comprises an amino acid sequence that is long enough tomake the tag accessible for binding by a binding molecule bound on asolid matrix, and is flexible enough to freely expose the tag forcapture by the binding molecule. Hence, a peptide linker used toseparate the tag and the recombinant HIV-1 gp140 may be flexible orrigid. In some embodiment, a peptide linker is at least 20 amino acidsin length. In some embodiment, a peptide linker may have 23 amino acidsin length. In some embodiment, a peptide linker is longer than 23 aminoacids in length. The nucleic acid sequence for a peptide linker may varycorresponding to the peptide linker selected. In some embodiments, theamino acid sequence of the linker contains repeats of glycine as well asamino acids such as alanine and serine, and proline. In someembodiments, a peptide linker may have an amino acid sequence of SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, inwhich the peptide linker comprising SEQ ID NO: 8 is an HRV-3C flexiblelinker, and the peptide linker comprising SEQ ID NO: 9 is an HRV-3crigid linker. In some embodiment, a peptide linker may be a foldonsequence (27aa) that has an amino acid sequence comprises SEQ ID. NO: 1.

A nucleic acid sequence of a tag may also vary according to need. Forexample, a nucleic acid sequence of a tag may encode a STREP-TAG® IIcomprising SEQ ID NO: 2, an octa-histidine tag comprising SEQ ID NO: 3,or a hexa-histidine tag comprising SEQ ID NO: 4.

Correspondingly, the recombinant DNA in some embodiment encompasses anucleic acid sequence encoding a recombinant HIV-1 gp140 comprising agp120 and a gp41 ectodomain, in which the gp120 and the gp41 ectodomainare joined together by a junction sequence containing a furin cleavagesite REKR (SEQ ID NO: 14). In some embodiments, the gp41 ectodomain istruncated at aa664 or a position not beyond aa664, based on HXB2numbering. In some embodiments, a nucleic acid sequence encoding a furincleavage site REKR (SEQ ID NO: 14) is mutated to encoding a sequence ofSEKS (SEQ ID NO: 15), resulting in producing a recombinant HIV-1 gp140that is furin cleavage resistant. In some embodiments, a nucleic acidsequence encoding a furin cleavage site REKR (SEQ ID NO: 14) is mutatedto encoding a sequence of RRRRRR (SEQ ID NO: 13), resulting in producinga recombinant HIV-1 gp140 that is furin cleavage proficient.

In some embodiments, a recombinant DNA has a nucleic acid sequenceencoding a recombinant HIV-1 gp140 that has “SOSIP” mutations comprisingA501C, T605C, and I559P. In some embodiments, the recombinant DNA has asequence encoding a recombinant HIV-1 gp140 that has both “SOSIP”mutations and “stabilizing” mutations. The “stabilizing” mutationsinclude I535M, Q543L, S553N, K567Q, and R588G.

According to one embodiment of the present invention, a nucleic acidsequence encoding a recombinant protein comprising a recombinant HIV-1gp140 fused to a tag through a linker disclosed herein may be derivedfrom any HIV-1 clade sequence. For example, a recombinant DNA may have anucleic acid sequence encoding a recombinant protein engineered based ona sequence of an HIV-1 clade B JRFL gp140 or HIV-1 clade B SF162 gp140.In another embodiment, a recombinant DNA may have a nucleic acidsequence encoding a recombinant protein engineered based on a sequenceof an HIV-1 clade A BG505 gp140. In one embodiment, a recombinant DNAhas a nucleic acid sequence encoding a recombinant protein comprisingSEQ ID NO: 10. In one embodiment, a recombinant DNA has a nucleic acidsequence encoding a recombinant protein comprising SEQ ID NO: 11. In oneembodiment, a recombinant DNA has a nucleic acid sequence encoding arecombinant protein comprising SEQ ID NO: 12.

Embodiments further provide various vectors for expression therecombinant proteins comprising recombinant HIV-1 gp140 fused to a tagthrough a peptide linker disclosed herein (may be called tagged HIV-1gp140). A vector may have a regulatory region operably linked to anucleic acid sequence encoding a recombinant protein described above.The regulatory region regulates the expression of the recombinantprotein in a cell carrying the vector. Once being expressed in cellscarrying the vector and secreted into culture medium that the cellsgrow, the recombinant protein has a capacity of assembly intorecombinant trimers mimicking native HIV-1 Env trimers. In someembodiment, the expression of the recombinant is regulated under aregulatory region comprising an inducible promoter, and the recombinantprotein is not consistently expressed in a cell carrying the vector butcan be induced as needed. In some embodiment, the expression of arecombinant protein described herein in a cell carrying the vector iscontrolled under a constitutively promoter. A cell carrying a vectorcomprising a constitutive promoter such as a CMV promoter willconstitutively express the tagged recombinant HIV-1 gp140. In someembodiments, a human cytomegalovirus (CMV) promoter locates at upstreamof the nucleic acid sequence encoding a recombinant protein comprising arecombinant HIV-1 gp140 attached to a tag via a peptide linker. CMVpromoter is a constitutive promoter and is always active.

Embodiments further provide various vectors comprising expressioncassette to construct expression vectors for producing recombinantprotein described herein. In some embodiments, a vector comprising anexpression cassette may comprise a regulatory region operably linked toa first nucleic acid sequence that encodes a secretion signal peptide.The first nucleic acid sequence is linked to a second nucleic acidsequence with an insertion region comprising two or more restrictionsites. The insertion region is use for insertion a recombinant DNAencoding a recombinant HIV-1 gp140. The second nucleic acid sequenceencodes a peptide linker and a tag. As a result, a vector to express arecombinant protein described herein may be easily constructed byinsertion a recombinant nucleic acid sequence encoding a recombinantHIV-1 gp140 using the two restriction sites in the insertion region. Arecombinant nucleic acid sequence encoding a recombinant HIV-1 gp140 canbe engineered by either overlap-extension PCR or gene assembly PCR usingappropriated set of primers, and by modification of any HIV-1 cladegp140.

In some embodiments, the insertion region has two restriction sites:NheI and NotI. In some embodiments, three alanines are located atN-terminus of the linker. In some embodiments, the sequence encoding asecretion signal peptide is a sequence encoding a human CD5 signalpeptide. A promoter in the vector may be a constitutive promoter or aninducible promoter. A promoter may be a CMV promoter. In someembodiments, the vector is a plasmid vector and can be transformed intobacteria to store or to amplify, and can be transfected into mammaliancells to express the recombinant protein. The recombinant protein hasthe capacity to form recombinant trimers mimicking native HIV-1 enveloptrimers. Once expressed in cells, the recombinant protein can besecreted into cell culture medium and assemble into recombinant trimers.

FIG. 3 illustrates an example of a vector that comprises an expressioncassette and can be used for insertion of a recombinant DNA encoding arecombinant HIV-1 gp140 and expression a recombinant HIV-1 gp140 fusedto a tag via a peptide linker. As shown in FIG. 3, a vector 300 maycontain a CD5 secretion signal, a peptide linker, and various tags suchas STREP-TAG® II and octa-histidine tags (SEQ ID NO: 3) described inPanel A of FIG. 2. In some embodiments, a peptide linker contains threealanines at the N-terminus of the linker (which is called “alaninelinker”). In one example, an insertion region 320 between the CD5 signaland the alanine linker contains two or more restriction sites. The twoor more restriction sites may be restriction sites NheI and NotI. Theseplasmid vector DNAs may be isolated from 5-alpha competent E. coli cellsand be digested with NheI and NotI and dephosphorylated with alkalinephosphatase. Recombinant DNA encoding a recombinant HIV-1 gp140 may beinserted into the vector at the restriction sites NheI and NotI.

Embodiments provide methods to purify recombinant trimers describedherein. According to embodiments, the material used to capture arecombinant HIV-1 envelope protein gp140 and recombinant trimers may bea binding molecule bound or immobilized on a solid matrix such asagarose beads. Binding molecules may be SREP-TACTIN®, nickel, etc.

In some embodiments, purification of the recombinant proteins or trimersmay be carried out by centrifugation. In some embodiments, purificationof the recombinant proteins or trimers is carried out by affinitychromatography and by size exclusion chromatography (SEC), and the sizeexclusion chromatography is conducted after the affinity chromatographyis conducted.

In some embodiments of the present invention, purification is achievedby column chromatography. By this approach, a binding molecule bound ona solid matrix is packed onto a column, and a culture medium containingthe recombinant protein or the recombinant trimer runs through thecolumn to allow the recombinant protein or the recombinant trimer bindto the binding molecule. Subsequently, a wash buffer runs through thecolumn and an elution buffer is applied to the column to elute therecombinant protein or the recombinant trimer from the solid matrix.

Alternatively, in some embodiments, purification of the recombinantprotein or the recombinant trimer is done by using a batch treatment. Abinding molecule bound and immobilized on a solid matrix is added into aculture medium containing the recombinant protein or the recombinanttrimer in a container. After mixing, the recombinant protein or therecombinant trimer binds to the binding molecule and can be separatedfrom the culture medium by centrifugation. For example, STREP-TACTIN®beads may be added to a culture medium containing recombinant trimerscomprising STREP-TAG® to form a bead mixture allowing the bindingbetween the STREP-TACTIN® beads and the recombinant trimers. TheSTREP-TACTIN® beads bound to the recombinant trimers may be thenseparated from the culture medium by centrifugation. Bounded trimers mayfurther be eluted with elution buffer. Alternatively, the STREP-TACTIN®beads bound to the recombinant trimers or the recombinant proteins maybe packed onto a column, followed with washing and elution on the columnpacked with the STREP-TACTIN® beads to purify the recombinant trimers orthe recombinant proteins.

Embodiments of the present invention demonstrate that the linkerapproach is highly effective to directly capture a recombinant proteincomprising a recombinant HIV-1 gp140 from a culture medium. In someembodiments, STREP-TACTIN® beads are directly added to a cell culturemedium into which the recombinant protein is secreted. The beads thatbind the recombinant protein may be separated by simple centrifugation.The recombinant protein bound on the beads may be then eluted using mildbuffer conditions such as conditions containing desthiobiotin.Similarly, in some embodiments, the recombinant HIV-1 gp140 attached toan octa-histidine tag (SEQ ID NO: 3) with a long linker in the middle iscaptured by Nickel beads. The recombinant HIV-1 gp140 bound on theNickel beads is eluted with imidazole. In some embodiments, the linkeris effective when it is ˜23 amino acids or longer and both a flexiblelinker and a rigid linker are equally effective in capturing arecombinant gp140. In some embodiments, a linker comprises a foldonsequence of SEQ ID NO: 7. The foldon sequence has a length of 27 aminoacids.

In some embodiments, the recombinant HIV-1 gp140 for producing therecombinant trimers comprises three “SOSIP” mutations (in comparing witha native HIV-1 gp140). The three “SOSIP” mutations include A501C, T605C,and I559P. In some embodiments, a recombinant HIV-1 gp140 may furtherencompass five “stabilizing” mutations (in comparing with a native HIV-1gp140). The five “stabilizing” mutations include I535M, Q543L, S553N,K567Q, and R588G.

According to embodiments, a variety of recombinant gp140 variants,cleaved, uncleaved, partially glycosylated, fully glycosylated, fromclades A and B, are purified by this approach. Upon purification andoptimization, recombinant trimers that mimic native HIV-1 Env trimersare obtained. Since the structure of the HIV-1 trimer base is conservedamong different HIV viruses, this approach can be broadly applied toproduce recombinant gp140 and recombinant trimers from any HIV-1 cladeor strain, wherein the recombinant trimers mimic native trimers ofvarious HIV-1 clade or strains.

Therefore, embodiments of the present invention develop a new and simpleapproach to produce HIV-1 envelope trimers. In some embodiments, theC-terminus of gp140 is attached to a tag such as STREP-TAG® II with along linker separating the tag from the massive trimer base andglycan-shield. This allows a capture of near-homogenous gp140 directlyfrom the supernatant of culture medium. Extensive biochemicalcharacterizations show that cleavage of gp140 is not essential fortrimerization, but it triggers a conformational change that channelstrimers into correct glycosylation pathways generating compactthree-blade propeller-shaped trimers. Uncleaved trimers enter aberrantpathways resulting in hyper-glycosylation, nonspecific crosslinking, andconformational heterogeneity. Even the cleaved trimers showmicro-heterogeneity in gp41 glycosylation.

Embodiments of the present invention are further defined in thefollowing examples. It should be understood that these examples aregiven by way of illustration only. From the above discussion and theseexamples, one skilled in the art can ascertain the essentialcharacteristics of embodiments of the present invention. Withoutdeparting from the spirit and scope thereof, one skilled in the art canmake various changes and modifications of the invention to adapt it tovarious usages and conditions. Although specific terms have beenemployed herein, such terms are intended in a descriptive sense and notfor purposes of limitation. All publications, including patents andnon-patent literature, referred to in this specification are expresslyincorporated by reference herein.

EXAMPLES Example 1

Experimental Procedures

Antibodies

The following reagents are obtained through the NIH AIDS ReagentProgram, Division of AIDS, NIAID, HIV-1 gp120 Monoclonal Antibody (2G12)^(36, 37, 38, 39, 40) from Dr. Hermann Katinger, HIV-1 gp120 MAb(VRC01)¹⁷ from Dr. John Mascola, PGT 121 (Cat#12343),⁴¹ HIV-1 gp41Monoclonal Antibody (F240)⁴² and HIV-1 gp120 Monoclonal Antibody(F105)^(43, 44, 45, 46) from Dr. Marshall Posner and Dr. Lisa Cavacini.The PG9,¹⁹ PG16,¹⁹ PGT145,⁴¹ PGT151,⁴⁷ and b6,¹⁶ are obtained fromScripps Research Institute and International AIDS Vaccine InitiativeNeutralizing Antibody Center (IAVI NAC). Polyclonal Bas against HIV-1JREL gp140 are raised in mice in our laboratory.

Clone Constructions

The furin-expressing plasmid, Furin: FLAG/pGEM7Zf(+), is obtained fromDr. Gary Thomas (Vollum Institute, Portland, Oreg.). The furin fragmentfrom this plasmid is sub-cloned into pcDNA3.1(−) (Life Technologies)using EcoRI and HindIII restriction sites.

Codon optimized gp140 DNAs from JRFL-FD, SF162-FD, and CONPEP-FD areprovided by Dr. Peter Kwong (Vaccine Research Center, NIH). These DNAscontained the sequence corresponding to gp120 and gp41 ectodomain up toaa683. In addition, they have human CD5 secretion signal at the 5′end,furin cleavage resistant mutation SEKS (SEQ ID NO: 15) at the junctionof gp120 and gp41, and bacteriophage T4 fibritin trimerization motiffollowed by the hexa-histidine tag (SEQ ID NO: 4) at the C-terminus.⁴⁸Using the JRFL-FD as the starting template, a series of additionalmutations are introduced. These include, for instance, SOSIPmutations,^(49,50) stabilizing mutations,⁵¹ enhanced furin cleavage siteRRRRRR (SEQ ID NO: 13),⁵² and various truncations shown in FIG. 2 and inthe following examples. JRFL gp120 clone is also constructed from thesame template by polymerase chain reaction (PCR) amplification of theappropriate sequence corresponding to gp120.

BG505 (BG505.W6M.ENV.C2)^(28,53) gp140 envelope sequence iscodon-optimized and the optimized sequence is synthesized using theGenArt Strings technology (Life Technologies). During this process, aseries of mutations are also introduced as follows: Asn at aa332 tointroduce an N-linked glycosylation site that allows binding of BG505gp140 to 2G12 BnAb;⁵⁴ SOSIP;²⁸ RRRRRR (SEQ ID NO: 13);⁵² and variousother mutations described in the following examples.

A series of modified pcDNA3.1(−) vectors are constructed, eachcontaining CD5 secretion signal, a liker containing three alanines, andvarious Strep-Tag II and Octa-histidine tags (SEQ ID NO: 3) described inthe following examples. Restriction sites NheI and NotI are introducedin between the CD5 signal and the alanine linker. These plasmid vectorDNAs isolated from 5-alpha competent E. coli cells (New England BioLabs,Inc.) are digested with NheI and NotI and dephosphorylated with FastAPalkaline phosphatase (Life Technologies).

The gp140 (and gp120) clones (recombinant DNA encoding recombinant HIV-1gp140) are constructed by either overlap-extension PCR⁵⁵ or geneassembly PCR⁵⁶ using appropriate sets of primers. Restriction sites forNheI and NotI are introduced into the end primers. The amplified DNAsare digested with NheI and NotI and purified by agarose gelelectrophoresis. The DNAs are then ligated with the NheI-NotI-digestedand dephosphorylated pcDNA3.1(−) plasmid DNA. Directional insertion ofgp140 DNA results in the in-frame fusion of gp140 with CD5 signalpeptide at the N-terminus and the alanine linker followed by varioustags at the C-terminus (see FIG. 2, Panel B).

The gp140 (and gp120) clones are transformed into 5-alpha competent E.coli cells (New England BioLabs, Inc) and the plasmid DNAs are purifiedusing the GeneJET plasmid miniprep kit (Life Technologies). The DNAs arethen sequenced to confirm 100% accuracy of the cloned gp140 DNA. Fortransfection into mammalian cells, the plasmid DNAs are purified usingthe GeneJET plasmid midiprep kit (Life Technologies) as per themanufacturer's instructions.

Small-Scale Transfection.

Suspension cells HEK293F (Life Technologies) and HEK293S GnTI⁻ (ATCCCRL-3022) are maintained in FreeStyle 293 expression medium (LifeTechnologies). The cells are incubated in a Multitron Pro shaker (InforsHT) at 37° C. in 8% CO₂. In the case of HEK293S GnTI⁻, the growth mediumis supplemented with 1% heat-inactivated fetal bovine serum (FBS,Quality Biologicals). For transfection, cells are grown overnight to adensity of 1×10⁶ cells per ml. Two hours prior to transfection, 6 mlcultures are centrifuged at 100 g for 5 min and resuspended in freshmedium to a density of 3×10⁶ cells per ml in the absence of FBS. Threeml of cells are then transferred to each well of a 16.8 ml 6 Well ClearNot Treated plates (Corning Inc.). For CR (and gp120) DNA, 6 μg of gp140plasmid DNA is added to the cells followed by the addition of linearpolyethylenimine (PEI25k, Polyscience Inc.) to a PEI:DNA (wt/wt) ratioof 3:1. For CP DNA, the cells are cotransfected with 3 μg of furinplasmid DNA. The cells are then incubated at 37° C. in 8% CO₂ whileshaking at 130 r.p.m. overnight. After 12 h, 2 ml of fresh medium, 1 mlHyClone SFM4HEK293 medium (GE Healthcare), and protein expressionenhancing sodium butyrate⁵⁷ (SIGMA-ALDRICH) to a final concentration of2 nM are added to the cells. On day 5, the supernatant is harvested andclarified using a 0.2 μm filter (Corning Inc.).

Large-Scale Transfection

Transfection is carried out similar to the small scale transfection, butit is scaled up to 1.2 L cultures in a 2.8 L flask and incubated at 37°C. in 8% CO₂ while shaking at 90 r.p.m.

Small-Scale Gp140 Purification

To inactivate biotin present in the supernatant, 20 μl of Bio-Lockbiotin blocking solution (iba Life Sciences) is added to 5 ml of thesupernatant containing the secreted gp140 (or gp120). After 30 minincubation at 4° C., 100 μl of STREP-TACTIN® beads (Qiagen) are addedand allowed to rotate overnight at 4° C. The bead mixture is spun downat 200 r.p.m. to pellet the beads. The beads are then applied to a spincolumn (PIERCE™) and briefly centrifuged to remove residual supernatantand then washed twice with 50 mM Tris-HCl, pH 8, and 300 mM NaCl. Thebound gp140 or gp120 proteins are eluted with 200 μl of STREP-TACTIN®elution buffer (2.5 mM d-Desthiobiotin (SIGMA), 25 mM Tris-HCl, pH 8,and 150 mM NaCl).

Large-Scale Gp140 Purification

To prevent nonspecific protease degradation, protease inhibitor tablets(Roche Diagnostics) are added to the clarified supernatant according tomanufacturer's instructions. To inactivate free biotin present in theculture medium, BioLock-biotin blocking solution (iba Life Sciences) isadded and the medium is incubated at 4° C. for 30 min. The gp140 ispurified by STREP-TACTIN® affinity chromatography followed by sizeexclusion chromatography (SEC). The supernatants are loaded onto a 1 mlSTREP-TACTIN® column (Qiagen) at 0.7 ml/min using the ÄKTA prime-plusliquid chromatography system (GE Healthcare). Nonspecifically boundproteins are washed off by passing at least 20 column volumes of washbuffer (50 mM Tris-HCl, pH 8, and 300 mM NaCl) until the absorbancereaches baseline level. The Strep-Tagged gp140 proteins are then elutedwith elution buffer (2.5 mM d-Desthiobiotin (SIGMA), 25 mM Tris-HCl pH 8and 150 mM NaCl) at a flow rate of 1 ml/min. The peak fractions arepooled and concentrated using 100 kDa MWCO Amicon Ultra-4 centrifugalfilter units (Millipore). The samples are then applied to a Hi-Load16/600 Superdex-200 (prep-grade) size exclusion column (GE Healthcare)equilibrated with the gel filtration buffer (25 mM Tris-HCl, pH 8, 150mM NaCl). Chromatography is done using the ÄKTA FPLC system (GEHealthcare) and fractions are collected and stored in 10% glycerol at−80° C.

The gp140 clones fused to hexa-histidine (SEQ ID NO: 4) orocta-histidine tags (SEQ ID NO: 3) are purified by HisTrap affinitychromatography followed by SEC. The culture supernatant is loaded onto a1 ml HisTrap HP column (GE Healthcare) at a flow rate of 0.7 ml/minusing the ÄKTA prime-plus liquid chromatography system (GE Healthcare).Nonspecifically bound proteins are removed using a buffer containing 50mM Tris-HCl, pH 8, 300 mM NaCl and 20 mM imidazole until the absorbancereached baseline level. The proteins are then eluted using a 20-500 mMimidazole gradient. The peak fractions are then applied to Hi-Load16/600 Superdex-200 (prep-grade) size exclusion column (GE Healthcare)and purified as described above.

SDS-PAGE and Blue Native (BN) PAGE

SDS-PAGE analyses are performed using 4-20% gradient Tris-glycine gels(Life Technologies) or home-made 10% gels in the presence (reducing) orabsence (non-reducing) of DTT. The BLUEstain™ protein ladder 11-245 kDa(a three-color protein standard with prestained proteins covering a widerange molecular weights produce by Gold Biotechnology®) is used as amolecular weight (MW) marker. BN-PAGE is performed using the Novex®NativePAGE™ Bis-Tris gel system in 4-16% gradient gels according tomanufacturer's instructions (Life Technologies). In the case of JRFL-FD,a native 4-12% gradient Tris-Glycine gel (Life Technologies) is usedwith Tris-Glycine buffer (Bio-Rad). The NativeMark™ unstained proteinstandard (Life Technology) is used as the MW marker. All gels arestained with Coomassie blue R-250 solution.

Protease Cleavage

SEC-purified gp140 trimers are incubated with 10-fold serial dilutions(1-0.001 μg/ml) of Proteinase K (Thermo Scientific) at 37° C. for 1 h.The same preparation incubated at 4° C. and 37° C. without protease isused as a negative control. The samples are electrophoresed on reducingSDS gels for cleavage resistant gp140 and non-reducing SDS gels forcleavage proficient gp140.

Deglycosylation

For STREP-TACTIN® purified gp140, 1 μl (500 Units) of PNGase F (NewEngland BioLabs, Inc.) is used to deglycosylate 10 μg of protein in theabsence of DTT following manufacturer's instructions. For SEC-purifiedtrimers, deglycosylation is performed under native conditions using 3 μl(1,500 Units) of PNGase F per 10 μg of protein and by incubating for 5 hat room temperature.

STREP-TACTIN® ELISA

STREP-TACTIN® coated microplates (iba Life Sciences) are coated with 1μg/ml SEC-purified gp140 trimers in a volume of 100 μl per well ofbuffer (25 mM Tris-HCl pH 7.6, 2 mM EDTA, and 140 mM NaCl) and incubatedfor 2 h at room temperature. Following 3 washes with PBST (0.05%Tween-20 in PBS), 100 μl of serially diluted antibodies (10-0.001 μg/ml)in PBS are added to the wells and the plates are incubated for 1 h at37° C. After 3 washes with PBST, the plates are incubated with 100 μl ofrabbit anti-human antibody (Santa Cruz Biotechnology) diluted 1:3,000 inPBS for 30 min at 37° C. After the final 3 washes with PBST, thereaction is developed with peroxidase (TMB Microwell PeroxidaseSubstrate system, KPL). The reaction is terminated by adding 100 μl ofBlueSTOP solution (KPL) and OD₆₅₀ is recorded using VersaMax™ ELISAMicroplate Reader (Molecular Devices).

Western Blotting

Polyclonal mouse antibodies against HIV-1 gp140 prepared in ourlaboratory are used as the primary antibody and rabbit HRP-conjugatedanti-mouse IgG (H+L) is used as the secondary antibody (Novex, LifeTechnologies). For STREP-TAG® II detection, StrepMAB-Classic HRPconjugated antibody (iba Life Sciences, dilution 1:1,000 in PBS) isused. Band intensities are measured using Biorad Gel doc XR+System andImage Lab software.

Negative-Stain EM

Samples are diluted to 20-30 μg/ml and added to a glow-dischargedcarbon-coated grid. Samples are left on the grid for 2 min, blotted witha filter paper, and stained with Nano-W™ (Nanoprobes, Yaphank, N.Y.) for30 s, with two cycles of rinsing followed by stain application. Afterthe last round of staining, the grid is blotted and allowed to drycompletely before being imaged. Grids are imaged on an FEI Tecnai T12microscope operating at 120 kV. Images are captured at a nominalmagnification of 67,000× on a Gatan UltraScan CCD using a dose of 20electrons per Angstrom squared. Particles are selectedsemi-automatically using e2boxer within EMAN2 with a box width of 200Angstroms. Reference-free 2D class averages are generated using EMAN2.⁵⁸Briefly, several particles are manually picked to initiate automatedparticle picking using e2boxer within EMAN2. After automated particlepicking, reference-free 2D class averages are generated using e2refine2dwithin EMAN2. Each sample goes through 15 iterations of 2Dclassification and 32 classes are generated per sample.

Example 2

Conventional Strategies have not been Very Effective to Produce HIV-IEnv Trimers

This example shows that a STREP-TAG® II with an extended linker allowsrapid purification of HIV-1 gp140 envelope trimers.

Several codon-optimized gp140 constructs from HIV-1 strains JRFL, SF162(clade B viruses), and CONPEP (clade C) for production of Env trimersare tested. As an example, FIG. 4 is a schematic image of the JRFLfoldon-gp140 recombinant construct showing the positions of the cleavageresistant SEKS mutation (SEQ ID NO: 15), foldon, and His-tag. The gp140DNA containing gp120 and gp41 ectodomain sequences truncated at aa664 or683 (HXB2 numbering) is cloned under the control of the CMV promoter(FIG. 4) and transfected into a variety of mammalian cell lines (293F,293T, 293EXPI, CHO, GNTI⁻). With a signal peptide fused to theN-terminus, gp140 is secreted into the culture medium and the efficiencyof production is quantified. Both cleavage resistant (CR) and cleavageproficient (CP) clones are tested. For CR gp140, the furin cleavage siteREKR (SEQ ID NO: 14) between gp120 and gp41 is mutated to SEKS (SEQ IDNO: 15) and for CP gp140, it is mutated to RRRRRR (SEQ ID NO: 13) andco-transfected with a second furin-containing plasmid to enhancecleavage.⁵²

Of the three signal peptides tested, CD5, tPA, and Gluc, CD5 showconsistently better expression. Fusing a hexa-histidine (His) tag (SEQID NO: 4) at the C-terminus of recombinant gp140 did not affectexpression, whereas an N-terminal tag shows poor expression probablybecause the hydrophilic tag affected cleavage of the adjacent, largelyhydrophobic, signal peptide. However, the C-terminal tag bound poorly,if at all, to Ni-agarose.

In some example, clones are also constructed by inserting the 27aa phageT4 fibritin trimerization motif (foldon)⁴⁸ which has a sequencecomprising SEQ ID NO: 7 between the C-terminus of recombinant gp140 andthe His-tag (e.g., JRFL, FIG. 3). As shown in FIG. 4, for CR gp140, thefurin cleavage site REKR (SEQ ID NO: 14) between gp120 and gp41 ismutated to SEKS (SEQ ID NO: 15). These constructions produce a mixtureof trimers and higher oligomers but no protomers (monomers and dimers).These trimers bind to Ni-agarose and can be further purified by sizeexclusion chromatography (SEC), which resolves the oligomers, but theelution profiles overlap (FIGS. 5, 6, and 7). These results areconsistent with the foldon-based trimers reported by otherinvestigators^(26,59,60) (also see Table 1 below).

FIG. 5 shows elution profile of trimers and oligomers on SEC forpurification of uncleaved JRFL gp140 foldon trimers. FIG. 6 is an imageof native gel of starting material from HisTrap column that is loaded onSEC (lane S). The gel is stained with Coomassie blue. Lanes “M” show MWmarkers. The MWs in kDa of the marker proteins are shown on the left.FIG. 7 is an image of native gel of the SEC fractions. The gels in FIG.6 and FIG. 7 are stained with Coomassie blue. Lanes “M” show MW markers.The MWs in kDa of the marker proteins are shown on the left. Furtherbiochemical analyses show that the protomers of these trimers arenonspecifically crosslinked with disulfide bonds (see below).

Recombinants without any tag are constructed and recombinant gp140 iscaptured using lectin beads (Galanthus nivalis lectin or concanavalinA). The protein is then purified by SEC. But the yields of the trimersvary either by direct application of the culture supernatant or after2-3× concentration by tangential flow filtration. Aggregation of some ofthe gp140 during lectin chromatography occurs. Furthermore, the bindingpotency of lectin diminishes progressively after each use.

FIG. 8 illustrates negative-stain EM of the peak trimer fraction fromFIG. 5. FIG. 9 illustrates 2D class averages of foldon trimers shown inFIG. 8. As illustrated in FIGS. 7 and 8, negative-stain EM of trimersproduced by the above approaches shows heterogeneous mixtures ofparticles. Relatively few are classic three-blade propeller shaped, andsome, for instance the JRFL foldon trimers, show variably shapedparticles 810 (FIG. 8) and 910 (FIG. 9), similar to that reported byGeorgiev et al,⁶⁰ even though all these preparations behave as “true”trimers by SEC and Blue native (BN) gel electrophoresis (See Table 1).

In Table 1, various approaches used for the purification of HIV-1trimers are compared. As shown in table 1, the approach of fusing aneight amino acid STREP-TAG® II tag through a >20 amino acid linker tothe C-terminus of gp140 provides several useful features and is broadlyapplicable to generate trimers from potentially any HIV-1 virus.

TABLE 1 Comparison of various approaches used for the purification ofHIV-1 trimers Lectin 2G12 Foldon STREP-TAG ® II Ligand Galanthus nivalisBnAb 2G12 coupled Ni (or a heavy metal Modified lectin attached to toSepharose 4B such as Co) attached streptavidin, solid matrix (Sanders2013) to solid matrix referred to as STREP-TACTIN ®, attached to solidmatrix gp140 Wild-type Wild-type The 27-amino acid The 8-amino acidtrimerization motif, STREP-TAG ® II referred to as foldon peptide witha >20 or fibritin, followed amino acid linker by His-tag fused to fusedto the C- the C-terminus of terminus of gp140 gp140 Specificity Low,broad High, narrow High High All molecules (not Requires epitope Bindsto hexa- or Binds to STREP- only proteins) containing α1-2-octa-histidine tag TAG ® II at the C- containing mannose linked mannosesin (SEQ ID NOS: 4 terminus captured appropriate and 3) at the structuralcontext; C-terminus envelope proteins of variant HIV viruses lacking theepitope may not be captured; these must be mutated to create a bindingsite for 2G12 BnAb (Murin 2014 & Sanders 2013) Elution Strong reagentStrong reagent Strong reagent Mild reagent (1M mannose) (3M MgCl₂)(200-400 mM (2.5 mM d- imidazole) Desthiobiotin) Purity Low to MediumHigh Low to Medium High In addition to Captures only the In addition toHis- Because of high gp120, gp140 envelope protein tag containingspecificity to protomers molecules gp140 protomers STREP-TACTIN ®,(monomers and containing the 2G12 (monomers and only the STREP- dimers)and trimers, epitope; these dimers) and trimers, TAG ® containingmannose-containing include gp120, Ni can non- gp140 protomers non-targetgp140 protomers specifically bind to (monomers and molecules will also(monomers and non-target dimers) and trimers be captured and co- dimers)and trimers contaminating will be captured purified; the proteinspurified sample therefore may contain significant amounts of thesecontaminants End No No Yes Yes specificity Does not Does not Sincebinding Since binding discriminate discriminate requires the C- requiresthe C- between the full- between the full- terminally fused tag,terminally fused tag, length gp140 length gp140 the purified sample thepurified sample molecules and molecules and essentially containsessentially contains gp120 or truncated gp120 or truncated full-lengthgp140 full-length gp140 products of gp140; products of gp140; molecules;gp120 molecules; gp120 the latter would the latter would and most of theand most of the remain as remain as truncated products truncatedproducts contaminants contaminants will be excluded by will be excludedby the column the column Quality of Medium High Low High trimers Theco-purified High percentage Primarily used for High percentagecontaminants might (>90%) of three- production of (>90%) of three-affect the quality of blade propeller uncleaved trimers; blade propellerthe trimers. shaped trimers trimers are irregular shaped trimersdemonstrated with shaped and non- demonstrated with cleaved BG505specifically cleaved JRFL gp140 gp140 crosslinked Stripping No No YesYes Contaminants Stripping with urea Can be stripped Can be strippednonspecifically or NaOH will likely with EDTA, urea, or with NaOHmultiple bound to the column disrupt epitope NaOH multiple times cannotbe binding; washing times completely stripped with 3M MgCl₂ off usingmight remove some conventional of the contaminants reagents such as ureaor NaOH as these will denature the lectin; washing with high saltbuffers can strip some of the contaminants Re-use Limited Not known HighHigh Can be re-used but Can be stripped and Can be stripped and thebinding capacity re-used multiple re-used multiple significantly timeswithout times without reduced after each significantly losingsignificantly losing use the binding the binding capacity. capacity.Capacity Low to Medium Not known High High 3 mg ligand/ml 50 mgligand/ml 9 mg ligand/ml Cost Medium High Low Low $130 for 2 ml or $565for 1 mg of $23 for 2 ml of Ni- $166 for 2 ml of 6 mg of Galanthus 2G12Ab (Polymun NTA Agarose STREP-TACTIN ® nivalis lectin GNL Scientific)(Qiagen) Superflow Plus (Vector Cost might be Since complete (Qiagen)Laboratories) higher because, i) stripping can be Since complete Costwould be all the 2G12 may done, the same stripping can be higher thanthe not be coupled to column can be re- done, the same amount shownSepharose in an used for purification column can be re- because activeform; and ii) of a different gp140 used for purification independentindependent protein; cross of a different gp140 columns may need columnsmay need contamination, if protein; cross to be used for to be used forany, would be contamination, if purification of purification of minimalany, would be different gp140 different gp140 minimal proteins to avoidproteins to avoid cross-contamination cross-contamination

Example 3

Extended STREP-TAG® II Allows Efficient Isolation of Gp140

This example illustrates an approach that allows selective capture ofgp140 directly from the culture medium and is desirable for theproduction of trimers. Previous attempts to achieve this by fusing gp140with a tag such as the His-tag have failed. It is hypothesized thatthese failures stemmed from the possibility that the tag, when attachedto the base of the gp140 structure, is probably occluded, a problemfurther compounded by the presence of glycan shield, with up to 12glycans attached to the C-terminal heptad repeat (HR)-2 helices. If thehypothesis is correct, extending the tag away from the base should makeit more accessible for binding. This reasoning is supported by anexample showing that an insertion of a 27aa foldon sequence comprisingSEQ ID NO: 7 between the C-terminus of gp140 and the His-tag allowsefficient binding to Ni-agarose.

A series of 36 recombinant clones are constructed as shown in FIG. 2 byfusing the gp140 C-terminus to STREP-TAG® II and octa-histidine tag (SEQID NO: 3) with various linkers in the middle. STREP-TAG® II is an Baapeptide (WSHPQFEK) comprising SEQ ID NO: [[1]]2 that binds to modifiedstreptavidin, namely STREP-TACTIN®, at μM affinity and stringentspecificity. Yet, the complex can be dissociated with desthiobiotin, amild condition. Clade B JRFL gp140 is chosen as a template to evaluatethis approach, but it is also constructed, in parallel, clade A BG505gp140 clones for comparison. Three “SOSIP” mutations and five“stabilizing” mutations are introduced to stabilize JRFLtrimers.^(49, 50) The three “SOSIP” mutations include A501C, T605C, andI559P mutations. The A501C and T605C mutations create an intra-protomerdisulfide bond between gp120 and gp41, and the I559P mutation in theheptad repeats HR1 helix strengthens inter-subunit (gp41) interactions.The five “stabilizing” mutations in or near HR1 (I535M, Q543L, S553N,K567Q, and R588G) strengthen gp120 and gp41 interactions at theinterface.^(49,50,51)

FIGS. 10 and 11 are images of reducing SDS polyacrylamide gels showingprotein patterns of the samples as indicated at the top according to oneembodiment of the present invention. Gels are stained with Coomassieblue. Lanes labeled as “M” show MW markers. The MWs in kDa of the markerproteins are shown on the left. The data demonstrate that the STREP-TAG®II approach is highly effective to capture gp140 from the culturemedium. Strep-Tagged gp140 with a short (Ala)₃ (or GlySerGlySer (SEQ IDNO: 17)) linker bound poorly to STREP-TACTIN® (FIG. 10, lane 3), whereasthe Twin STREP-TAG® containing 23aa linker is efficiently captured (FIG.10, lane 4), even though both clones expressed gp140 at similar levels(FIG. 10, lanes 1 and 2).

As shown in FIG. 11, bound gp140 can be specifically dissociated with2.5 mM desthiobiotin and the eluted protein is ˜95% pure (e.g., FIG. 11,lanes 1 and 2). An HRV 3C protease cleavage site engineered between thegp140 C-terminus and the linker is not cleaved, consistent with thehypothesis that a large protease molecule would encounter clashes withthe protomer base. Various forms of gp140 can be efficiently captured:uncleaved (FIG. 11, lanes 1-8) or cleaved (FIG. 11, lanes 9-16);truncated at aa664 (FIG. 11, lanes 1-4 and 9-12) or aa683 (FIG. 11,lanes 5-8 and 13-16); tagged with octa-His (SEQ ID NO: 3) with aflexible linker (FIG. 11, lanes 4, 8, 12, 16) or a rigid linker (FIG.11, lanes 3, 7, 11, 15).

Various forms of gp140 developed from clade A(BG505), clade B (JRFL),and clade A-E viruses can also be captured. FIG. 12 is an image of Bluenative (BN) gel of STREP-TACTIN® purified gp140 samples. Lanes labeledas “M” show MW markers. The MWs in kDa of the marker proteins are shownon the left. Gels are stained with Coomassie blue. As shown in FIG. 12,cleaved and uncleaved gp140 from clade A and clade B viruses can becaptured. In addition, trimers from SF162 (clade B) and 40007 (cladeCRF01 A-E) viruses are also purified. Furthermore, unlike the capturemethods employing lectin or 2G12 BnAb, the approach disclosed hereinspecifically captures full-length gp140 molecules and excludes gp120 andthe truncated molecules that are often generated by nonspecificproteases (see Table 1).

Example 4

Strep-Tagged JRFL Gp140 Produces Abundant Amounts of Trimers

CP and CR gp140 produce cleaved and uncleaved trimers, respectively.FIG. 13 is an image of SDS gel of samples under reducing (+DTT) ornon-reducing (−DTT) conditions. Short arrows show oligomers of gp120 orgp140 formed by nonspecific disulfide crosslinking. Long arrowscorrespond to the ladder of gp41 ectodomain bands glycosylated tovarying extents. Lanes labeled as “M” show MW markers. The MWs in kDa ofthe marker proteins are shown on the left. Gels are stained withCoomassie blue. As shown in FIG. 13, cleavage by furin is nearlycomplete in CP gp140 (FIG. 13, lane 5) whereas little or no cleavage isevident in CR gp140 (FIG. 13, lane 3). The cleaved gp120 and gp41subunits are covalently associated through the SOS disulfide bond asevident from the appearance of a single 140 kDa band under non-reducingconditions (FIG. 13, lane 6) and two bands (gp120 and gp41) underreducing conditions (FIG. 13, lane 5). However, a ladder of five gp41bands is also seen (arrows in lane 5, FIG. 13), probably correspondingto glycosylation of 0 to 4 N-linked glycosylation sites (see below). TheCR gp140, on the other hand, shows a single 140-kDa band under bothreducing and non-reducing conditions (FIG. 13, lanes 3 and 4). The lackof cleavage of CR gp140 is further confirmed by Western blotting using ahighly sensitive STREP-TAG® specific mAb as shown in FIG. 14.

Whether the CR gp140 also forms the SOS bond cannot be determined.Varying levels of higher oligomers are also seen in all preparations(including gp120), probably due to nonspecific disulfide crosslinking ofthe protomers under non-reducing conditions, but much less so with thecleaved gp140 (arrows in lanes 2, 4, and 6 of FIG. 13). About two-thirdsof the Strep-Tagged JRFL CP664-gp140 assemble into trimers (FIG. 12,lane 1), whereas CR664-gp140 produces more dimers than trimers (FIG. 12,lane 2). Similar patterns are also seen with Strep-Tagged BG505 gp140.However BG505 produces higher levels of uncleaved trimers (FIG. 12, lane4, compare with lane 2), and the trimer bands are more diffused thanJRFL indicating more extensive glycosylation as also evidenced byslightly higher MW of these bands (FIG. 12, lanes 3 and 4, compare withlanes 1 and 2).

Example 5

Truncation of Cleaved gp140 Beyond aa664 Results in Poor Gp140Production

This example illustrates that truncations beyond aa664 produce little orno gp140 trimers. (FIGS. 15, 16, 17, 18 and 19). In this example, arapid strategy to optimize various parameters for maximal trimerproduction, using any HIV-1 Env sequence, is developed.

FIG. 15 shows an exemplary screening strategy 1500 to optimizerecombinant various parameters for maximal trimer production, using anyHIV-1 Env sequence. More than 40 different Strep-Tagged gp140 clones1522 are constructed at step 1520. Each clone 1522 encompasses anexpression cassette to express human CD5 secretion signal peptide 1524fused Strep-Tagged gp140 protein 1526. Human CD5 secretion signalpeptide 1524 is fused at N-terminus of Strep-Tagged gp140 protein 1526.Linker 1527 and STREP-TAG® II 1528 are located at C-terminus ofStrep-Tagged gp140 protein 1526. Each clone is transfected into a smallvolume (6 ml) of cells at step 1530. At step 1550, the efficiency ofgp140 expression (FIG. 16) and the efficiency of cleavage (FIG. 17) areanalyzed by directly testing the culture medium. In addition, thesecreted gp140 is captured by STREP-TACTIN® beads and further probed forcleavage and gp41 glycosylation (FIG. 18) and trimer formation (FIG.19). Different parameters tested include: point of truncation,importance of SOSIP mutations and cleavage, production in 293F or GnTI⁻cells (GnTI⁻ cells lack N-acetylglucosaminetransferase 1 and cannotintroduce complex glycosylations), and clade B (JRFL) or A (BG505) gp140(an N-glycosylation site is introduced in BG505 at aa332 to make itequivalent to JRFL gp140).⁶¹ Results are shown in FIGS. 16, 17, 18 and19.

FIGS. 16, 17, 8, and 19 illustrate that truncations beyond aa664 producelittle or no gp140 trimers. FIG. 16 is an image of a non-reducing SDSgel comparing the production of uncleaved and cleaved gp140 in theculture medium for aa truncations at aa664 and aa683. FIG. 17 is animage showing reducing SDS gel comparing the production of gp140recombinants truncated at various aa positions at the C-terminus. Endpoint numbers correspond to the aa at the end of the C-terminus. Thegp140 is captured by Strep-Tactin beads and further probed for cleavageand gp41 glycosylation (FIG. 18) and trimer formation (FIG. 19).Approximately the same amount of gp140 is loaded in each lane to comparethe aa664 and aa683 proteins. FIG. 18 is an image showing reducing SDSgel of uncleaved and cleaved gp140 proteins truncated at aa664 andaa683. Arrows in darker shade and lighter shade correspond todifferentially glycosylated gp41 ectodomain bands of gp140 proteinstruncated at aa664 and aa683, respectively. FIG. 19 is an image showingBN gel of STREP-TACTIN® purified gp140 samples. FIGS. 16, 18, and 19 areWestern blots using mouse anti-gp140 polyclonal antibody. FIG. 17 is aWestern blot using STREP-TAG® II specific mAb. Data shown are for GnTI⁻produced JRFL gp140.

Similar patterns are observed with both JRFL and BG505 gp140s expressedin 293F or GnTI⁻ cells. SOSIP mutations prove essential as without themmost of the protein aggregate and cannot be captured by STREP-TACTIN®.Unexpectedly, however, cleaved gp140 truncated beyond aa664 producelower amounts of gp140 in the culture medium (FIGS. 16 and 17). Theaa672 and aa683 constructs produce 3-5 times lower amount whereasfurther truncation results in near complete loss of gp140 production.This result is not due to poor cleavage because 683-gp140 is efficientlycleaved producing, as expected, slightly larger gp41 ladder bands (FIG.18, lane 2, compare with lane 1). In contrast, production of uncleaved683-gp140 is not significantly affected. Unlike the cleaved 683-gp140which is expressed at lower levels (FIG. 16, lanes 2 and 6; FIG. 10,compare lanes 9-12 with lanes 13-16), the expression of uncleaved683-gp140 is nearly as high as 664 (FIG. 16, lanes 3 and 7 vs 4 and 8).Finally, the aa683 protein shows a tendency to aggregate, as shown byits appearance largely as a high molecular weight (MW) smear in the BNgel (FIG. 19, lane 3).

The above results suggest that cleavage triggers a conformational changein the MPER, which may lead to exposure of some of the hydrophobicresidues leading to aggregation. This hypothesis is consistent with theprevious reports by Klasse et al., and Ringe et al.,²⁶ which shows thatthe cleaved aa681 (from KNH1144) and aa683 (from BG505) gp140 proteinsform micelles at the MPER, presumably through interaction of the exposedhydrophobic residues of MPER with the lipid components.

Example 6

Cleavage is Essential for Production of Authentic HIV-1 Trimers

This example illustrates that cleavage is essential for production ofauthentic HIV-1 trimers. FIGS. 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42 are imagesillustrating the purification of cleaved and uncleaved JRFL gp140trimers.

FIGS. 20, 21 and 22 are images showing single-step purification ofStrep-Tagged gp140 from the culture supernatant by STREP-TACTIN® column.FIG. 20 is an image of reducing SDS gel of various gp140 samples: Lane Sis culture supernatant; Lanes 1-6 are fractions eluted fromSTREP-TACTIN® column with 2.5 mM desthiobiotin. FIG. 21 is an imageshowing BN gel of STREP-TACTIN® purified gp140 that is loaded on SEC(lane S) and three major fractions eluted from SEC (lanes 1-3corresponding to the pooled peaks 1-3 shown in FIG. 21). FIG. 22 is animage showing typical elution profile of gp140 oligomers from Superdex200 size exclusion column (SEC).

As shown in FIG. 20, STREP-TACTIN® purified gp140 is ˜95% pure, but itcontains a mixture of trimers and protomers, as well as some highmolecular weight (MW) species (FIG. 21, lane S). SEC separates theseinto three major fractions (FIG. 22): (i) high MW fraction that elutesimmediately after the void volume and migrates as a diffused band on BNgel (FIG. 21, lane 1); (ii) trimers, which elutes as a relatively sharppeak and migrates as a compact band on BN gel (FIG. 21, lane 2); and(iii) two overlapping peaks of protomer dimers and monomers (FIG. 21,lane 3).

To determine which of the trimers are authentic; cleaved or uncleaved,293F-produced (complex glycans) or GnTI⁻-produced (high mannose, nocomplex glycans), trimers are expressed on a large scale (1-4 liters)and purified by STREP-TACTIN® capture and SEC. The yields of gp140 areas follows: 293F CR—˜20 mg/L; CP—˜12 mg/L; GnTI⁻ CR—˜3 mg/L; CP—˜1 mg/L.Each SEC fraction is then analyzed by SDS-PAGE under reducing conditionsto assess purity and cleavage, BN-PAGE to assess oligomeric state,negative EM to assess the shape of the trimer, and antigenicity toassess conformation (see below).

FIGS. 23, 24, 25, 26 and 27 are images showing the purification ofcleaved trimers expressed in 293F cells. FIGS. 28, 29, 30, 31 and 32 areimages showing the purification of cleaved trimers expressed in GNTI⁻cells. FIGS. 33, 34, 35, 36 and 37 are images showing the purificationof uncleaved trimers expressed in 293F cells. FIGS. 38, 39, 40, 41 and42 are images showing the purification of uncleaved trimers expressed inGNTI⁻ cells. FIGS. 23, 28, 33, and 38 are images of BN-PAGE gel of theSEC fractions. FIGS. 24, 29, 34, and 39 are images of reducing SDS gelof fractions corresponding to those showing in FIGS. 23, 28, 33, and 38.Each SEC fraction is analyzed by SDS-PAGE under reducing conditions toassess purity and cleavage in FIGS. 23, 28, 33, and 38. In FIGS. 23, 24,28, 29, 33, 34, 38, and 39, the gels are stained with Coomassie blue;lanes S represent starting material (STREP-TACTIN®-purified gp140)loaded on SEC; lanes M show MW markers; the MWs in kDa of markerproteins are shown on the left. FIGS. 25, 26, 30, 31, 35, 36, 37, 40,41, and 42 are images of negative-stain EM of the peak SEC fractions toassess the shape of the trimers. FIGS. 27 and 32 are images ofreference-free 2D class averages of trimers. Example trimers 2520, 2620,2720, 3020, 3120, 3220, 3520, 3620, 3720, 4020, 4120, and 4220 arerespectively shown in FIGS. 25, 26, 27, 30, 31, 32, 35, 36, 37, 40, 41,and 42.

As shown in FIGS. 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41 and 42, the trimers are purified to nearhomogeneity, up to 2-3 mg/L (CP trimers), as well as established thecriteria for their authenticity. First, in the case of JRFL Env, thefraction of gp140 recovered as trimers is 3-5 fold greater with cleavedgp140 than with uncleaved gp140 (compare lanes S, 5-7 of FIG. 23 withFIG. 33). Second, the uncleaved trimers are of poor quality whencompared to the cleaved trimers. Unlike the CP trimer fractions thatshow a sharp band on the BN gel (FIG. 23, lanes 3-7), the CR trimerfractions contain significant levels of diffused and high MW species(FIG. 33, lanes 3-7). The latter represents conformationallyheterogeneous molecules, as is also evident from their poor reactivitywith the conformation-specific BnAb PGT145 as shown in FIG. 43.

FIG. 43 is an exemplary graph illustrating conformational heterogeneityof uncleaved trimers produced in 293F cells. SEC fractions 2-5 from FIG.33 are coated on STREP-TACTIN® plates at a fixed protein concentrationof 1 μg/ml and ELISAs are performed using the BnAbs 2G12 (darker shadebars 4310) or PGT145 (lighter shade bars 4320). Inset 4340 shows theCoomassie blue stained BN gel of fractions 2-5 depicting the presence ofvarious amounts of smear in the fractions. The smear representsdifferential migration of conformationally heterogeneous trimers on thenative gel. Note the poor reactivity of fraction 2 containing anextensive smear to the conformation-specific PGT145 BnAb when comparedto fraction 5 with a lesser smear. On the other hand, the 2G12 BnAbwhich is not dependent on the conformation of the trimer reactedequivalently to both fractions 2 and 5. As shown in FIG. 43, thereactivity is the lowest with fractions containing the highest amount ofthese species.

Third, the protomers of the uncleaved trimers as well as thefoldon-trimers (also uncleaved) are nonspecifically crosslinked throughdisulfide bonds whereas the cleaved trimers show much less crosslinking.See FIG. 44.

FIG. 44 is a set of gel images showing that protomers of uncleavedtrimers are nonspecifically crosslinked with disulfide bonds. TheSEC-purified JRFL trimers; STREP-TAG® uncleaved (CR), foldon-uncleaved(FD), and STREP-TAG® cleaved trimers (CP) are electrophoresed undernon-reducing (Panel A of FIG. 44) or reducing (Panel B of FIG. 44)conditions without PNGase F treatment (lanes 1, 3, 5, 7, 9, 11) or withPNGase F treatment (lanes 2, 4, 6, 8, 10, 12). A ladder of high MW bandsare present in the FD uncleaved and STREP-TAG® uncleaved trimers (arrowsin lanes 1 and 3), but not in the cleaved trimers (lane 5). As shown byelectrophoresis under reducing conditions and treatment with PNGase F,these bands correspond to nonspecific disulfide crosslinked protomers,but not to differences in glycosylation. All the high MW bands areconverted to a single band under reducing conditions (lanes 7, 9), butthe ladder remains after PNGase F treatment (lanes 2, 4), although thedeglycosylated (DG) bands migrate faster due to the removal of glycans(when compared to lanes 1 and 3). The cleaved trimers do not show thehigh MW ladder bands under non-reducing conditions (lane 5) and convertto a faster-migrating DG band after PNGase F treatment (lane 6). Underreducing conditions, the cleaved trimers give rise to gp120 and a ladderof gp41 bands (lane 11), and faster-migrating DG gp120 and single DGgp41 band (lane 12) after PNGase F treatment.

Fourth, the uncleaved trimers are more susceptible to nonspecificproteolysis, as evidenced by greater proteolysis of the CR trimers byproteinase K than the CP trimers (FIG. 45). FIG. 45 is a set of imageshowing proteinase K sensitivity of cleaved and uncleaved JRFL trimers.Panel A of FIG. 45 shows SEC-purified trimers from 293F or GnTI− cellsbeing treated with Proteinase K at the indicated concentrations for 1 hrat 37° C. and electrophoresed on a reducing SDS gel followed byCoomassie blue staining. The 37° C. lane corresponds to control sampleincubated at 37° C. for 1 hr without Proteinase K. Note that theuncleaved trimers are more susceptible to proteolysis than the cleavedtrimers, and also that the 293F trimers are more susceptible toproteolysis than the GnTI− trimers. Panel B of FIG. 45 illustratesDensitometric Quantification of the undigested gp140 bands from panel Aof FIG. 45.

Finally, negative-stain EM shows that the cleaved trimers appear asthree-blade propeller-shaped particles (FIGS. 25, 26, 30, and 31,reference-free 2D class averages are shown in FIGS. 27 and 32) whereasthe CR fractions show fewer such particles and most are irregularlyshaped (FIGS. 35, 36, 37, 40, 41, and 42).

Overall, the above results are consistent with the behavior of theuncleaved and cleaved trimers generated by the 2G12 approach (see Table1).²⁶

Example 7

Uncleaved Trimers are Hyper-Glycosylated

This example illustrates that GnTI⁻ cells produce better quality trimersthan the 293F cells although the yields are lower in GNU⁻ cells.(compare FIG. 23 with FIG. 28, FIG. 24 with FIG. 29, FIG. 25 with FIG.30, FIG. 26 with FIG. 31, FIG. 27 with FIG. 32, FIG. 33 with FIG. 38,FIG. 34 with FIG. 39, FIG. 35 with FIG. 40, FIG. 36 with 41, and FIG. 37with FIG. 42). For instance, the diffused high MW species describedabove are not seen in the CR trimers produced by GnTI⁻ cells (compareFIG. 33 with FIG. 38, lanes 1-7). Negative-stain EM shows a highernumber of propeller-shaped trimers in the GnTI⁻-produced CR trimers thanin the 293-produced trimers (compare FIGS. 35, 36, and 37 with FIGS. 40,41, and 42, respectively), which, in part, is due to heterogeneity inglycosylation. GnTI⁻ cells predominantly add Man5GlcNAc2 which isfurther processed by complex glycosylation in 293F cells. Presence ofSTREP-TAG® II at the C-terminus of gp41 allows evaluation ofglycosylation using Strep-Tag-specific mAbs.

FIGS. 46, 47, and 48 are exemplary images showing that the uncleavedtrimers produced in 293F cells are hyper-glycosylated. FIG. 46 is animage of western blot of reducing SDS gel using the STREP-TAG® mAbshowing the ladder of gp41 ectodomain bands. Lanes “M” show MW markers.The MWs in kDa of marker proteins are shown on the left. The gel isstained with Coomassie blue. FIG. 47 shows densitometric quantificationof the intensity of the gp41 ladder bands shown in FIG. 46. FIG. 48 isan image of a Western blot of reducing SDS gel using STREP-TAG® IIspecific mAb.

The presence of STREP-TAG® II at the C-terminus of gp41 allowsevaluation of glycosylation status by using STREP-TAG® II specific mAbs.As shown in FIGS. 46 and 47, a ladder of five gp41 bands appears whenCPgp140 trimers are electrophoresed under reducing conditions. Of thesebands, band #3 shows maximum intensity (FIG. 46 and FIG. 47). Since theJRFL gp41 ectodomain contains a cluster of four predicted N-linkedglycosylation sites near its C-terminus, these bands probably correspondto glycosylation of 0 to 4 sites. This is confirmed by deglycosylationwith PNGase F, which converts the ladder to a single species thatmigrates at the same position as the lowest band in the laddercorresponding to the unglycosylated gp41 (FIG. 48, lanes 2,4,6,8).Although a similar pattern is observed in both 293F and GnTI⁻ cells, thefully glycosylated 293F-gp41 bands are more diffused than the same fromGNTI⁻-gp41 (FIG. 48, compare lanes 1 to 3 and 5 to 7), presumably due tocomplex-glycosylation. BG505 CP-gp140 shows similar banding patternsexcept that it appeared to undergo more extensive glycosylation. Theseresults demonstrate “micro-heterogeneity” in gp41 glycosylation, albeitto a higher extent in 293F cells than in GnTI⁻ cells. Heterogeneity ofgp41 glycosylation was also inferred in previous reports.^(62, 63)

The heterogeneity is even more severe with the uncleaved trimers.Indeed, the uncleaved trimers produced by 293F cells are“hyper-glycosylated”. FIG. 49 is an image of Coomassie blue-stainednon-reducing SDS gel of purified uncleaved (CR) and cleaved (CP) JRFLand BG505 gp140 trimers produced in 293F or GnTI⁻ cells. FIG. 50 is animage of Coomassie blue-stained non-reducing SDS gel of samples fromFIG. 49 after treatment with PNGase F. Lanes “M” show MW markers. TheMWs in kDa of marker proteins are shown on the left. As shown in FIG.49, under non-reducing conditions, both the CP gp140 and CR gp140migrate at the same position when gp140 is produced by the GnTI⁻ cells(FIG. 49, lanes 3 and 4). The 293F gp140 migrates slower than the GnTI⁻gp140 (compare lanes 1 and 3, FIG. 49), which is expected because gp140undergoes complex glycosylations in 293F cells. Unexpectedly, however,the 293F-produced CR gp140 migrates slower than CP gp140 (FIG. 49,compare lanes 1 and 2). Upon deglycosylation with PNGase F, all gp140bands, whether uncleaved or cleaved, produced in 293F or GnTI⁻ cells,migrate at the same position (FIG. 50). The same pattern is alsoobserved with the BG505 gp140 trimers tested in parallel (see BG505lanes in FIGS. 48 and 49). These results demonstrate that the 293Funcleaved trimers are hyper-glycosylated when compared to their cleavedcounterparts.

Example 8

Antigenic Signatures Discriminate Between Uncleaved and Cleaved Trimers

In this example, an ELISA platform that can differentiate the structuraland conformational states of cleaved and uncleaved trimers is used.

FIG. 51 is an image showing the epitope signatures recognized by variousantibodies (Abs) in the 3D context of a gp140 trimer structure 5100 (PDB4tvp; the model is generated by PyMol. 69.⁶⁹ These epitope signaturesare color coded and include amino acid residues as well as glycans. ASshown in FIG. 51, a gp140 trimer structure 5100 has several epitopesignatures that are targeted by various antibodies. For example,antibodies VRC01, b6, F105, etc. can bind at epitope signature 5110;epitope signature 5120 can be bound by antibodies PG9, PG16, PGT145,etc.; epitope signature 5140 can be bound by 2G12, PGT121, etc.; epitopesignature 5150 can be bound by antibody F240; epitope signature 5160 canbe bound by antibody PGT151.

FIG. 52 is a set of graphs of results of ELISA performed with purifiedcleaved and uncleaved gp140 trimers with various mAbs. Purified cleavedand uncleaved gp140 trimers are coated on STREP-TACTIN® plates throughthe C-terminal STREP-TAG® II and incubated with various antibodies(mAbs) that recognize different epitope signatures shown in FIG. 51.Various mAbs are shown in the top left corner of each graph in FIG. 52.ELISAs are performed. The protein concentration of the trimers per wellis kept constant at 1 μg/ml. Each graph shows the binding curve fromthree replicates at the indicated concentrations of the mAb. Curves inlighter shade correspond to cleaved trimers; Curves in darker shadecorrespond to uncleaved trimers. The P value as determined by theunpaired two-tailed t test is <0.05 for PGT151, PGT145, PG9 and PG16 at1 μg/ml of Ab. Repetition of ELISAs several times with independentlypurified trimers yield similar results. The results of the reactivity ofthe trimers tested to BnAbs 2G12, VRC01, and PGT121 are shown in FIG. 52in graph 5212, graph 5214, and graph 5216, respectively. The results ofthe reactivity of the trimers tested to BnAbs PGT151 and PGT145 areshown in FIG. 52 in graph 5222 and graph 5224, respectively. The resultsof the reactivity of the trimers tested to BnAbs PG9 and PG16 are shownin FIG. 52 in graph 5232 and graph 5234, respectively. The results ofthe reactivity of the trimers tested to BnAbs b6, F105, and F240 areshown in FIG. 52 in graph 5242, graph 5244, and graph 5246,respectively.

Since coating is done at neutral pH (unlike at pH 9 in traditionalELISAs), it cause minimal, if any, structural perturbation. Moreover,the trimers are immobilized at a defined point; therefore, allimmobilized molecules are exposed in a similar orientation, in some waysmimicking the Env spikes displayed on the HIV-1 virion. Finally, the23aa flexible linker should make the trimer more accessible to Abbinding.

The reactivity of the trimers to BnAbs 2G12, VRC01, and PGT121 is firsttested. 2G12 recognizes a discontinuous epitope consisting of 3 or 4high mannose glycans in the gp120 domain.^(64, 65) VRC01 is a potentBnAb that binds to the CD4bs and neutralizes >90% of the primary HIV-1isolates.¹⁷ PGT121 primarily recognizes the complex glycan attached toN332.⁶¹ Consistent with the published data that the conformationalepitopes recognized by these Abs are well-exposed in trimers as well asin gp120, both cleaved and uncleaved trimers react strongly, andequivalently, to these Abs (FIG. 51).^(23, 26, 28, 66, 67)

The BnAb PGT151 recognizes a conformational epitope containing aaresidues and glycans present at the interface of gp120 and gp41 that isbetter exposed in the cleaved trimers.^(68, 69) The results show thatthe cleaved gp140 trimers exhibit stronger reactivity to PGT151 than theuncleaved trimers (see graph 5222 of FIG. 52), suggesting that cleavedtrimers achieve native-like conformation.

The BnAbs PG16 and PG9 are quaternary Abs that neutralize 70-80% of theprimary HIV-1 viruses. The quaternary specificity stems from its longhammerhead-shaped CDR which asymmetrically interacts with the V1, V2,and V3 loops of two protomers from the same trimer. The contact regionsinclude, primarily, the V1V2 loop glycan N160 and N156/N173, and residueK168 of one protomer and glycans N160 and N197 (V3 loop) of the adjacentprotomer.^(29, 70, 71) PGT145 is also a quaternary BnAb, however lesswell characterized, and it, like PG9 and PG16, recognizes the N160 andN156/N173 glycans.^(41, 70) Consistent with the expectation that acompact trimer would react better with the quaternary Ab, the cleavedtrimers react more strongly with PG9, PG16, and PGT145 BnAbs than theuncleaved trimers (FIG. 52).

Finally, the reactivity of the trimers with the non-neutralizing Abs(NnAbs) b6, F105, and F240 is tested. F105 and b6 recognize an epitopethat includes CD4bs whereas F240 binds to the immunodominant loop ofgp41 (aa592-604).^(16,42,67,68,69) The CR and CP trimers as well asprotomers reacted similarly with these NnAbs, although the reactivitywith F240 is poor overall probably because its epitope is partiallyoccluded. (FIG. 51, epitope signature 5150).

Collectively, these data demonstrate that the trimers display antigenicsignatures that are consistent with their cleaved or uncleaved states.Differential reactivity with the quaternary epitopes provides the bestbenchmark to ascertain the antigenic signature of compact, native-liketrimers.

Discussion

The trimeric envelope spike of HIV-1 virion makes the first contact withthe host cell. It triggers fusion of viral and host membranes anddelivers the nucleocapsid core into the cell. Trimer-specific Abs candisable Env function and block transmission of HIV. Development of arecombinant trimer immunogen, therefore, is one of the highestpriorities in the hunt for an effective HIV vaccine.^(26,31,75) However,a myriad of variations reported in the literature leads to confusion andcontroversy, and none can be broadly applied to diverse strains of HIV.For instance, a procedure that produces native-like trimers from A-cladeBG505 by using 2G12 BnAb to capture gp140 is not as effective with theB-clade JRFL trimers.^(26,31) Hence, another procedure is developed inwhich lectin capture and negative selection by F105 NnAb is used topurify trimers.³¹ These Ab-based approaches have inherent limitationssince the epitope signatures may vary from one HIV clade to another. Infact, it is necessary to mutate the wild-type BG505 gp140 in order tocreate the 2G12 binding epitope and allow for its purification by the2G12 BnAb.^(26,66) and moreover, the Abs are not readily available,prohibitively expensive, and not practical for vaccine production.

Here, a new approach is developed that allows production of HIV Envtrimers from potentially any HIV-1 clade or strain. Systematic analysesare presented to optimize trimer production, and biochemicalcharacterizations to define the signatures of trimers.

A key feature of the approach disclosed herein is to selectively capturegp140 Env directly from the culture supernatant under mild conditionsthat cause minimal, if any, perturbation to the structure or oligomericstate of the protein. Attempts to achieve this using an affinity taghave thus far failed because the tag is not accessible for interactionwith its binding partner. In accordance with the recent X-raystructures, the C-terminal aa664 would not be accessible as it is thelast residue of the long HR2 helices that encircle the base of the gp140trimer.^(29,69) It is further shielded by as many as 12 glycansemanating from these helices.^(29,69) Therefore it is essential not onlyto incorporate an exquisitely specific STREP-TAG® II, but also toseparate the tag from the base by a >20aa-long linker. Thesemodifications avoid clashes with the trimer base and allow purificationof near homogeneous protein in a single step. A variety of gp140variants; cleaved, uncleaved, GnTI⁻ glycosylated, and 293F glycosylatedfrom clades A, B, and A-E viruses can be purified by this approach.

The Strep-Tagged gp140 proteins behaved similar to the native gp140. Forinstance, the CP gp140 is nearly completely cleaved to gp120 and gp41and the CR gp140 remained uncleaved. SOSIP mutations are essential;otherwise, most of the gp140 aggregate into a high MW fraction.Curiously, gp41 glycosylation is heterogeneous, showing five gp41 bandscorresponding to glycosylation of zero to four sites of the fourN-linked glycosylation sites clustered in or near the 34aa-long HR2helix. This micro-heterogeneity, which is observed in both JRFL andBG505 gp140, may reflect a competition between the rate of glycosylationand the rate of folding of this transiently exposed structural element.

The results show that cleavage is not essential for trimerization perse, but it is essential for maturation into propeller-shaped particles.Uncleaved gp140 produced such native-like articles but in fewer andvariable numbers. Maturation might involve two, probably sequential,events, conformational transition and complex glycosylation.⁷⁶ Acleavage-triggered conformational transition can be deduced from anumber of experiments. Truncated CP gp140 constructs beyond aa664, e.g.,aa683, produce 3-5 times lower amounts of gp140, whereas the sametruncation in CR background is not significantly affected, and much ofthe aa683 protein aggregate. Thus, conformation of MPER where theseresidues are located must be different in the cleaved and uncleavedstates. These results are consistent with the previous reports by Klasseet al., and Ringe et al.,²⁶ which showed that the cleaved aa681 andaa683 proteins formed micelles at the MPER. Perhaps some of the residuesin the hydrophobic-rich MPER are better exposed in the cleaved state andassociate with the membrane. Structural studies suggest that the MPERforms an L-shaped bent helix and the residues 675-683 contact the virionmembrane.⁷⁷

Secondly, cleaved trimers exhibited greater stability and are lesssusceptible to proteolysis than the uncleaved trimers suggesting thatcleavage renders the trimers more compact and less accessible toprotease. Finally, negative-stain EM shows compact, propeller-shapedtrimers in the cleaved state and irregularly shaped “blobs” in theuncleaved state, as is also observed by Ringe et al with the 2G12produced trimers (Table 1).²⁶

Careful analysis of glycosylation patterns show that cleavage channelstrimers into the correct glycosylation pathway. Without cleavage,trimers from both JRFL and BG505 enter an aberrant pathway resulting inhyper-glycosylation, which traps the trimers in a loosely associatedstate. Consequently, the uncleaved trimers including the foldon trimersproduced by 293F cells are conformationally heterogeneous,nonspecifically disulfide crosslinked, more susceptible to proteolysis,and irregularly shaped. Presence of a diffuse smear in the native gel,poor reactivity with the conformation-specific PGT145 BnAbs, andheterogeneity in gp41 complex glycosylations, provide further evidenceof this phenotype. Finally, the uncleaved trimers from GnTI⁻ cells whichare unable to carry out hyper-glycosylations show a higher percentage ofnative-like trimers, further underscoring the negative effects ofhyper-glycosylation.

The strong reactivity of the CR and CP trimers with the BnAbs 2G12,VRC01, and PGT121 confirms that the Strep-Tagged trimers have acorrectly folded gp120 and gp41 ectodomain exposing the respectiveconformational epitopes. Preferential reactivity of the cleaved trimerswith the PGT151 BnAb further confirms the integrity of theconformational epitope that emerges at the interface of gp120 and gp41following cleavage. Strong reactivity with cleaved trimers, but not withuncleaved trimers, of the quaternary BnAbs PG9 and PG16 demonstratesthat CP gp140 protomers assemble into correct quaternary structure.

Contrary to some reports that the CR trimers, but not the CP trimers,react with the NnAbs, both of CR and CP trimers react similarly with theNnAbs b6, F240, and F105.⁶⁶ Table 2 shows the reactivity of variouscleaved and uncleaved trimers with non-neutralizing antibodies usingdifferent assay platforms. The table shows the reactivity of variouscleaved (CP) and uncleaved (CR) trimer preparations with thenon-neutralizing Abs b6, F105, and F240, using different assay platformsreported in the literature. Scores are assigned based on a comparison ofthe reactivity of different gp140 constructs reported in the same figurefrom each publication. Different publications are grouped into one lineif the scores match. Reactivity scores: +++ high, ++ moderate, + weak,+/− above baseline, − negative. The reactivity is dependent on the assayplatform (compare the reactivity of BG505.SOSIP.R6.664 in ELISA vs SPRvs BLI) and the presence of SOSIP mutation [compare the reactivity ofBG505.SOSIP.SEKS.664 (line 4) vs BG505.WT.SEKS.664 by SPR (line 5)].Also, the ELISA data of BG505.SOSIP.R6.664 is compared toBG505.WT.SEKS.664 but not to its counterpart BG505.SOSIP.SEKS.664.

TABLE 2 Reactivity of various cleaved and uncleaved trimers with non-neutralizing antibodies using different assay platforms: ConstructComposition Cleavage b6 F105 F240 References ELISA 1. BG505.SOSIP.R6.664Trimer CP +++ + ++ (26, 28) 2. BG505.WT.SEKS.664 Trimer CR +++ +++ +++(26) SPR 3. BG505.SOSIP.R6.664 Trimer CP −/+ NR − (26, 28, 66) 4.BG505.SOSIP.SEKS.664 Trimer CR + NR −/+ (26, 66) 5. BG505.WT.SEKS.664Trimer CR +++ NR +++ (26, 66) Bio-layer interferometry BLI 6.BG505.SOSIP.R6.664 Trimer CP ++ − NR (69) 7. JRFL.SOSIP.R6.663 Trimer CP+++ ++ NR (31)* 8. 16055.SOSIP.R6.663 Trimer CP +++ ++ NR (31)* NR—notreported *Before negative selection

Careful examination of the published reports, however, shows that thereactivity depended on the type of assay platform used, and thesequences of the CR and CP trimers compared are not identical. On theother hand, data illustrated in some examples are generated usingidentical CP and CR sequences (except for the cleavage site) and theSTREP-TACTIN® based ELISA platform is not expected to introducesignificant structural perturbations into the trimeric antigens.Furthermore, the HIV Env trimer is a dynamic structure and likelyoscillates between “closed” and “open” states, allowing the NnAb tointeract with the trimer when it opens transiently.^(78,79) Thus, strongreactivity to the quaternary-specific BnAbs such as PG9 and PG16 is themost reliable benchmark to assess the authenticity of the native-liketrimers.

In conclusion, a new system to produce, optimize, and characterize pureand native-like HIV-1 Env trimers is developed herein. Both cleavage andproper glycosylation are critical to generate compact, three-bladepropeller shaped particles, whereas without cleavage, the trimers areheterogeneous in conformation, nonspecifically crosslinked, andhyper-glycosylated, properties consistent with their irregular shape.The GnTI⁻ cells produced better quality trimers than the 293F cells.However, the 293F trimers might better recapitulate the native structurebecause GnTI⁻ cells lack complex glycosylations. The caveat, however, isthat the glycan structures introduced by the 293F cells are not know andif these glycan structures are the same as that present on the HIV-1virion. Micro-heterogeneity of glycosylations might also be a concern.Three criteria, namely 95% cleavage, near 100% propeller-shapedparticles, and strong reactivity to quaternary BnAbs, define authenticHIV-1 trimers. The approach disclosed herein provides several usefulfeatures (see TABLE 1) and is broadly applicable to generate trimersfrom potentially any HIV-1 virus for basic research as well as for humanclinical trials and vaccine manufacture. The well-behaved JRFL trimersdescribed here may serve as a good scaffold for further engineering togenerate a trimeric immunogen that can elicit transmission-blocking Absagainst diverse HIV-1 strains.

It is intended that the invention not be limited to the particularembodiment disclosed herein contemplated for carrying out thisinvention, but that the invention will include all embodiments fallingwithin the scope of the claims. For example, although the HIV-1 clade BJRFL gp140 and clade A BG505 gp140 sequences are used as templates inconstructing recombinant HIV-1 gp140 and in producing trimeric envelopespike that mimics native-like HIV-1 trimers, it will be appreciated thatsequences from other HIV-1 clades can be used as templates forproducing, purifying and testing trimers produced according to theapproach disclosed herein. Antibodies against HIV-1 that can be used incapturing “native-like” trimers are not limited to BnAb 2G12. Inaddition, tags that fused to recombinant HIV-1 gp140 are not limited toocta-histidine (SEQ ID NO: 3) and STREP-TAG® II.

REFERENCES

The following references are referred to above and are incorporatedherein by reference:

-   1. Kwong, P. D., and Mascola, J. R. (2012) Human antibodies that    neutralize HIV-1: identification, structures, and B cell ontogenies.    Immunity 37, 412-425-   2. Mascola, J. R., and Nabel, G. J. (2001) Vaccines for the    prevention of HIV-1 disease. Curr Opin Immunol 13, 489-495-   3. Wyatt, R., and Sodroski, J. (1998) The HIV-1 envelope    glycoproteins: fusogens, antigens, and immunogens. Science 280,    1884-1888-   4. Ward, A. B., and Wilson, I. A. (2015) Insights into the trimeric    HIV-1 envelope glycoprotein structure. Trends Biochem Sci-   5. Wilen, C. B., Tilton, J. C., and Doms, R. W. (2012) Molecular    mechanisms of HIV entry. Adv Exp Med Biol 726, 223-242-   6. Arthos, J., Cicala, C., Martinelli, E., Macleod, K., Van Ryk, D.,    Wei, D., Xiao, Z., Veenstra, T. D., Conrad, T. P., Lempicki, R. A.,    McLaughlin, S., Pascuccio, M., Gopaul, R., McNally, J., Cruz, C. C.,    Censoplano, N., Chung, E., Reitano, K. N., Kottilil, S., Goode, D.    J., and Fauci, A. S. (2008) HIV-1 envelope protein binds to and    signals through integrin alpha4beta7, the gut mucosal homing    receptor for peripheral T cells. Nat Immunol 9, 301-309 7. Cicala,    C., Arthos, J., and Fauci, A. S. (2011) HIV-1 envelope, integrins    and co-receptor use in mucosal transmission of HIV. J Transl Med 9    Suppl 1, S2-   8. Dalgleish, A. G., Beverley, P. C., Clapham, P. R., Crawford, D.    H., Greaves, M. F., and Weiss, R. A. (1984) The CD4 (T4) antigen is    an essential component of the receptor for the AIDS retrovirus.    Nature 312, 763-767-   9. McDougal, J. S., Nicholson, J. K., Cross, G. D., Cort, S. P.,    Kennedy, M. S., and Mawle, A. C. (1986) Binding of the human    retrovirus HTLV-III/LAV/ARV/HIV to the CD4 (T4) molecule:    conformation dependence, epitope mapping, antibody inhibition, and    potential for idiotypic mimicry. J Immunol 137, 2937-2944-   10. Klatzmann, D., Champagne, E., Chamaret, S., Gruest, J., Guetard,    D., Hercend, T., Gluckman, J. C., and Montagnier, L. (1984)    T-lymphocyte T4 molecule behaves as the receptor for human    retrovirus LAV. Nature 312, 767-768-   11. Alkhatib, G., Combadiere, C., Broder, C. C., Feng, Y.,    Kennedy, P. E., Murphy, P. M., and Berger, E. A. (1996) CC CKRS: a    RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for    macrophage-tropic HIV-1. Science 272, 1955-1958-   12. Feng, Y., Broder, C. C., Kennedy, P. E., and    Berger, E. A. (1996) HIV-1 entry cofactor: functional cDNA cloning    of a seven-transmembrane, G protein-coupled receptor. Science 272,    872-877-   13. Lederman, M. M., Penn-Nicholson, A., Cho, M., and    Mosier, D. (2006) Biology of CCR5 and its role in HIV infection and    treatment. JAMA 296, 815-826-   14. Furuta, R. A., Wild, C. T., Weng, Y., and Weiss, C. D. (1998)    Capture of an early fusion-active conformation of HIV-1 gp41. Nat    Struct Biol 5, 276-279-   15. Gallo, S. A., Finnegan, C. M., Viard, M., Raviv, Y., Dimitrov,    A., Rawat, S. S., Puri, A., Durell, S., and Blumenthal, R. (2003)    The HIV Env-mediated fusion reaction. Biochim Biophys Acta 1614,    36-50-   16. Roben, P., Moore, J. P., Thali, M., Sodroski, J., Barbas, C. F.,    3rd, and Burton, D. R. (1994) Recognition properties of a panel of    human recombinant Fab fragments to the CD4 binding site of gp120    that show differing abilities to neutralize human immunodeficiency    virus type 1. J Virol 68, 4821-4828-   17. Wu, X., Yang, Z. Y., Li, Y., Hogerkorp, C. M., Schief, W. R.,    Seaman, M. S., Zhou, T., Schmidt, S. D., Wu, L., Xu, L., Longo, N.    S., McKee, K., O'Dell, S., Louder, M. K., Wycuff, D. L., Feng, Y.,    Nason, M., Doria-Rose, N., Connors, M., Kwong, P. D., Roederer, M.,    Wyatt, R. T., Nabel, G. J., and Mascola, J. R. (2010) Rational    design of envelope identifies broadly neutralizing human monoclonal    antibodies to HIV-1. Science 329, 856-861-   18. Zwick, M. B., Jensen, R., Church, S., Wang, M., Stiegler, G.,    Kunert, R., Katinger, H., and Burton, D. R. (2005) Anti-human    immunodeficiency virus type 1 (HIV-1) antibodies 2F5 and 4E10    require surprisingly few crucial residues in the membrane-proximal    external region of glycoprotein gp41 to neutralize HIV-1. J Virol    79, 1252-1261-   19. Walker, L. M., Phogat, S. K., Chan-Hui, P. Y., Wagner, D.,    Phung, P., Goss, J. L., Wrin, T., Simek, M. D., Fling, S.,    Mitcham, J. L., Lehrman, J. K., Priddy, F. H., Olsen, 0. A.,    Frey, S. M., Hammond, P. W., Protocol, G. P. I., Kaminsky, S., Zamb,    T., Moyle, M., Koff, W. C., Poignard, P., and Burton, D. R. (2009)    Broad and potent neutralizing antibodies from an African donor    reveal a new HIV-1 vaccine target. Science 326, 285-289-   20. Benjelloun, F., Lawrence, P., Verrier, B., Genin, C., and    Paul, S. (2012) Role of human immunodeficiency virus type 1 envelope    structure in the induction of broadly neutralizing antibodies. J    Virol 86, 13152-13163-   21. Yu, L., and Guan, Y. (2014) Immunologic Basis for Long HCDR3s in    Broadly Neutralizing Antibodies Against HIV-1. Front Immunol 5, 250-   22. Esparza, J. (2013) A brief history of the global effort to    develop a preventive HIV vaccine. Vaccine 31, 3502-3518-   23. Chakrabarti, B. K., Feng, Y., Sharma, S. K., McKee, K., Karlsson    Hedestam, G. B., Labranche, C. C., Montefiori, D. C., Mascola, J.    R., and Wyatt, R. T. (2013) Robust neutralizing antibodies elicited    by HIV-1 JRFL envelope glycoprotein trimers in nonhuman primates. J    Virol 87, 13239-13251-   24. Kovacs, J. M., Nkolola, J. P., Peng, H., Cheung, A., Perry, J.,    Miller, C. A., Seaman, M. S., Barouch, D. H., and Chen, B. (2012)    HIV-1 envelope trimer elicits more potent neutralizing antibody    responses than monomeric gp120. Proc Natl Acad Sci USA 109,    12111-12116-   25. Nkolola, J. P., Cheung, A., Perry, J. R., Carter, D., Reed, S.,    Schuitemaker, H., Pau, M. G., Seaman, M. S., Chen, B., and    Barouch, D. H. (2014) Comparison of multiple adjuvants on the    stability and immunogenicity of a clade C HIV-1 gp140 trimer.    Vaccine 32, 2109-2116-   26. Ringe, R. P., Sanders, R. W., Yasmeen, A., Kim, H. J., Lee, J.    H., Cupo, A., Korzun, J., Derking, R., van Montfort, T., Julien, J.    P., Wilson, I. A., Klasse, P. J., Ward, A. B., and    Moore, J. P. (2013) Cleavage strongly influences whether soluble    HIV-1 envelope glycoprotein trimers adopt a native-like    conformation. Proc Natl Acad Sci USA 110, 18256-18261-   27. Liao, H. X., Lynch, R., Zhou, T., Gao, F., Alam, S. M., Boyd, S.    D., Fire, A. Z., Roskin, K. M., Schramm, C. A., Zhang, Z., Zhu, J.,    Shapiro, L., Program, N. C. S., Mullikin, J. C., Gnanakaran, S.,    Hraber, P., Wiehe, K., Kelsoe, G., Yang, G., Xia, S. M.,    Montefiori, D. C., Parks, R., Lloyd, K. E., Scearce, R. M.,    Soderberg, K. A., Cohen, M., Kamanga, G., Louder, M. K., Tran, L.    M., Chen, Y., Cai, F., Chen, S., Moquin, S., Du, X., Joyce, M. G.,    Srivatsan, S., Zhang, B., Zheng, A., Shaw, G. M., Hahn, B. H.,    Kepler, T. B., Korber, B. T., Kwong, P. D., Mascola, J. R., and    Haynes, B. F. (2013) Co-evolution of a broadly neutralizing HIV-1    antibody and founder virus. Nature 496, 469-476-   28. Sanders, R. W., Derking, R., Cupo, A., Julien, J. P., Yasmeen,    A., de Val, N., Kim, H. J., Blattner, C., de la Pena, A. T., Korzun,    J., Golabek, M., de Los Reyes, K., Ketas, T. J., van Gils, M. J.,    King, C. R., Wilson, I. A., Ward, A. B., Klasse, P. J., and    Moore, J. P. (2013) A next generation cleaved, soluble HIV-1 Env    Trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for    broadly neutralizing but not non-neutralizing antibodies. PLoS    Pathog 9, e1003618-   29. Julien, J. P., Cupo, A., Sok, D., Stanfield, R. L., Lyumkis, D.,    Deller, M. C., Klasse, P. J., Burton, D. R., Sanders, R. W.,    Moore, J. P., Ward, A. B., and Wilson, I. A. (2013) Crystal    structure of a soluble cleaved HIV-1 envelope trimer. Science 342,    1477-1483-   30. Lyumkis, D., Julien, J. P., de Val, N., Cupo, A., Potter, C. S.,    Klasse, P. J., Burton, D. R., Sanders, R. W., Moore, J. P.,    Carragher, B., Wilson, I. A., and Ward, A. B. (2013) Cryo-EM    structure of a fully glycosylated soluble cleaved HIV-1 envelope    trimer. Science 342, 1484-1490-   31. Guenaga, J., de Val, N., Tran, K., Feng, Y., Satchwell, K.,    Ward, A. B., and Wyatt, R. T. (2015) Well-Ordered Trimeric HIV-1    Subtype B and C Soluble Spike Mimetics Generated by Negative    Selection Display Native-like Properties. PLoS Pathog 11, e1004570-   32. Gao, G., Wieczorek, L., Peachman, K. K., Polonis, V. R.,    Alving, C. R., Rao, M., and Rao, V. B. (2013) Designing a soluble    near full-length HIV-1 gp41 trimer. J Biol Chem 288, 234-246-   33. Tao, P., Mahalingam, M., Marasa, B. S., Zhang, Z., Chopra, A.    K., and Rao, V. B. (2013) In vitro and in vivo delivery of genes and    proteins using the bacteriophage T4 DNA packaging machine. Proc Natl    Acad Sci USA 110, 5846-5851-   34. Sathaliyawala, T., Rao, M., Maclean, D. M., Birx, D. L.,    Alving, C. R., and Rao, V. B. (2006) Assembly of human    immunodeficiency virus (HIV) antigens on bacteriophage T4: a novel    in vitro approach to construct multicomponent HIV vaccines. J Virol    80, 7688-7698-   35. Liu, J., Bartesaghi, A., Borgnia, M. J., Sapiro, G., and    Subramaniam, S. (2008) Molecular architecture of native HIV-1 gp120    trimers. Nature 455, 109-113-   36. Buchacher, A., Predl, R., Strutzenberger, K., Steinfellner, W.,    Trkola, A., Purtscher, M., Gruber, G., Tauer, C., Steindl, F.,    Jungbauer, A., and et al. (1994) Generation of human monoclonal    antibodies against HIV-1 proteins; electrofusion and Epstein-Barr    virus transformation for peripheral blood lymphocyte    immortalization. AIDS Res Hum Retroviruses 10, 359-369-   37. Trkola, A., Purtscher, M., Muster, T., Ballaun, C., Buchacher,    A., Sullivan, N., Srinivasan, K., Sodroski, J., Moore, J. P., and    Katinger, H. (1996) Human monoclonal antibody 2G12 defines a    distinctive neutralization epitope on the gp120 glycoprotein of    human immunodeficiency virus type 1. J Virol 70, 1100-1108-   38. Mascola, J. R., Lewis, M. G., Stiegler, G., Harris, D.,    VanCott, T. C., Hayes, D., Louder, M. K., Brown, C. R., Sapan, C.    V., Frankel, S. S., Lu, Y., Robb, M. L., Katinger, H., and    Birx, D. L. (1999) Protection of Macaques against pathogenic    simian/human immunodeficiency virus 89.6PD by passive transfer of    neutralizing antibodies. J Virol 73, 4009-4018-   39. Etemad-Moghadam, B., Sun, Y., Nicholson, E. K., Karlsson, G. B.,    Schenten, D., and Sodroski, J. (1999) Determinants of neutralization    resistance in the envelope glycoproteins of a simianhuman    immunodeficiency virus passaged in vivo. J Virol 73, 8873-8879-   40. Crawford, J. M., Earl, P. L., Moss, B., Reimann, K. A.,    Wyand, M. S., Manson, K. H., Bilska, M., Zhou, J. T., Pauza, C. D.,    Parren, P. W., Burton, D. R., Sodroski, J. G., Letvin, N. L., and    Montefiori, D. C. (1999) Characterization of primary isolate-like    variants of simianhuman immunodeficiency virus. J Virol 73,    10199-10207-   41. Walker, L. M., Huber, M., Doores, K. J., Falkowska, E., Pejchal,    R., Julien, J. P., Wang, S. K., Ramos, A., Chan-Hui, P. Y., Moyle,    M., Mitcham, J. L., Hammond, P. W., Olsen, O. A., Phung, P., Fling,    S., Wong, C. H., Phogat, S., Wrin, T., Simek, M. D., Protocol, G. P.    I., Koff, W. C., Wilson, I. A., Burton, D. R., and    Poignard, P. (2011) Broad neutralization coverage of HIV by multiple    highly potent antibodies. Nature 477, 466-470-   42. Cavacini, L. A., Emes, C. L., Wisnewski, A. V., Power, J.,    Lewis, G., Montefiori, D., and Posner, M. R. (1998) Functional and    molecular characterization of human monoclonal antibody reactive    with the immunodominant region of HIV type 1 glycoprotein 41. AIDS    Res Hum Retroviruses 14, 1271-1280-   43. Posner, M. R., Elboim, H., and Santos, D. (1987) The    construction and use of a human-mouse myeloma analogue suitable for    the routine production of hybridomas secreting human monoclonal    antibodies. Hybridoma 6, 611-625-   44. Posner, M. R., Hideshima, T., Cannon, T., Mukherjee, M.,    Mayer, K. H., and Byrn, R. A. (1991) An IgG human monoclonal    antibody that reacts with HIV-1/GP120, inhibits virus binding to    cells, and neutralizes infection. J Immunol 146, 4325-4332-   45. Posner, M. R., Cavacini, L. A., Emes, C. L., Power, J., and    Byrn, R. (1993) Neutralization of HIV-1 by F105, a human monoclonal    antibody to the CD4 binding site of gp120. J Acquir Immune Defic    Syndr 6, 7-14-   46. Cavacini, L. A., Emes, C. L., Power, J., Underdahl, J.,    Goldstein, R., Mayer, K., and Posner, M. R. (1993) Loss of serum    antibodies to a conformational epitope of HIV-1/gp120 identified by    a human monoclonal antibody is associated with disease progression.    J Acquir Immune Defic Syndr 6, 1093-1102-   47. Falkowska, E., Le, K. M., Ramos, A., Doores, K. J., Lee, J. H.,    Blattner, C., Ramirez, A., Derking, R., van Gils, M. J., Liang, C.    H., McBride, R., von Bredow, B., Shivatare, S. S., Wu, C. Y.,    Chan-Hui, P. Y., Liu, Y., Feizi, T., Zwick, M. B., Koff, W. C.,    Seaman, M. S., Swiderek, K., Moore, J. P., Evans, D., Paulson, J.    C., Wong, C. H., Ward, A. B., Wilson, I. A., Sanders, R. W.,    Poignard, P., and Burton, D. R. (2014) Broadly neutralizing HIV    antibodies define a glycandependent epitope on the prefusion    conformation of gp41 on cleaved envelope trimers. Immunity 40,    657-668-   48. Yang, X., Lee, J., Mahony, E. M., Kwong, P. D., Wyatt, R., and    Sodroski, J. (2002) Highly stable trimers formed by human    immunodeficiency virus type 1 envelope glycoproteins fused with the    trimeric motif of T4 bacteriophage fibritin. J Virol 76, 4634-4642-   49. Binley, J. M., Sanders, R. W., Clas, B., Schuelke, N., Master,    A., Guo, Y., Kajumo, F., Anselma, D. J., Maddon, P. J., Olson, W.    C., and Moore, J. P. (2000) A recombinant human immunodeficiency    virus type 1 envelope glycoprotein complex stabilized by an    intermolecular disulfide bond between the gp120 and gp41 subunits is    an antigenic mimic of the trimeric virionassociated structure. J    Virol 74, 627-643-   50. Sanders, R. W., Vesanen, M., Schuelke, N., Master, A.,    Schiffner, L., Kalyanaraman, R., Paluch, M., Berkhout, B.,    Maddon, P. J., Olson, W. C., Lu, M., and Moore, J. P. (2002)    Stabilization of the soluble, cleaved, trimeric form of the envelope    glycoprotein complex of human immunodeficiency virus type 1. J Virol    76, 8875-8889-   51. Dey, A. K., David, K. B., Klasse, P. J., and Moore, J. P. (2007)    Specific amino acids in the Nterminus of the gp41 ectodomain    contribute to the stabilization of a soluble, cleaved gp140 envelope    glycoprotein from human immunodeficiency virus type 1. Virology 360,    199-208-   52. Binley, J. M., Sanders, R. W., Master, A., Cayanan, C. S.,    Wiley, C. L., Schiffner, L., Travis, B., Kuhmann, S., Burton, D. R.,    Hu, S. L., Olson, W. C., and Moore, J. P. (2002) Enhancing the    proteolytic maturation of human immunodeficiency virus type 1    envelope glycoproteins. J Virol 76, 2606-2616-   53. Hoffenberg, S., Powell, R., Carpov, A., Wagner, D., Wilson, A.,    Kosakovsky Pond, S., Lindsay, R., Arendt, H., Destefano, J., Phogat,    S., Poignard, P., Fling, S. P., Simek, M., Labranche, C.,    Montefiori, D., Wrin, T., Phung, P., Burton, D., Koff, W., King, C.    R., Parks, C. L., and Caulfield, M. J. (2013) Identification of an    HIV-1 clade A envelope that exhibits broad antigenicity and    neutralization sensitivity and elicits antibodies targeting three    distinct epitopes. J Virol 87, 5372-5383-   54. Kong, L., Lee, J. H., Doores, K. J., Murin, C. D., Julien, J.    P., McBride, R., Liu, Y., Marozsan, A., Cupo, A., Klasse, P. J.,    Hoffenberg, S., Caulfield, M., King, C. R., Hua, Y., Le, K. M.,    Khayat, R., Deller, M. C., Clayton, T., Tien, H., Feizi, T.,    Sanders, R. W., Paulson, J. C., Moore, J. P., Stanfield, R. L.,    Burton, D. R., Ward, A. B., and Wilson, I. A. (2013) Supersite of    immune vulnerability on the glycosylated face of HIV-1 envelope    glycoprotein gp120. Nat Struct Mol Biol 20, 796-803-   55. Higuchi, R., Krummel, B., and Saiki, R. K. (1988) A general    method of in vitro preparation and specific mutagenesis of DNA    fragments: study of protein and DNA interactions. Nucleic Acids Res    16, 7351-7367-   56. Stemmer, W. P., Crameri, A., Ha, K. D., Brennan, T. M., and    Heyneker, H. L. (1995) Single-step assembly of a gene and entire    plasmid from large numbers of oligodeoxyribonucleotides. Gene 164,    49-53-   57. Reeves, P. J., Kim, J. M., and Khorana, H. G. (2002) Structure    and function in rhodopsin: a tetracycline-inducible system in stable    mammalian cell lines for high-level expression of opsin mutants.    Proc Natl Acad Sci USA 99, 13413-13418-   58. Tang, G., Peng, L., Baldwin, P. R., Mann, D. S., Jiang, W.,    Rees, I., and Ludtke, S. J. (2007) EMAN2: an extensible image    processing suite for electron microscopy. J Struct Biol 157, 38-46-   59. Tran, K., Poulsen, C., Guenaga, J., de Val, N., Wilson, R.,    Sundling, C., Li, Y., Stanfield, R. L., Wilson, I. A., Ward, A. B.,    Karlsson Hedestam, G. B., and Wyatt, R. T. (2014) Vaccine-elicited    primate antibodies use a distinct approach to the HIV-1 primary    receptor binding site informing vaccine redesign. Proc Natl Acad Sci    USA 111, E738-747-   60. Georgiev, I. S., Joyce, M. G., Yang, Y., Sastry, M., Zhang, B.,    Baxa, U., Chen, R. E., Druz, A., Lees, C. R., Narpala, S., Schon,    A., Van Galen, J., Chuang, G. Y., Gorman, J., Harned, A., Pancera,    M., Stewart-Jones, G. B., Cheng, C., Freire, E., McDermott, A. B.,    Mascola, J. R., and Kwong, P. D. (2015) Single-chain soluble    BG505.SOSIP gp140 trimers as structural and antigenic mimics of    mature closed HIV-1 Env. J Virol-   61. Julien, J. P., Sok, D., Khayat, R., Lee, J. H., Doores, K. J.,    Walker, L. M., Ramos, A., Diwanji, D. C., Pejchal, R., Cupo, A.,    Katpally, U., Depetris, R. S., Stanfield, R. L., McBride, R.,    Marozsan, A. J., Paulson, J. C., Sanders, R. W., Moore, J. P.,    Burton, D. R., Poignard, P., Ward, A. B., and Wilson, I. A. (2013)    Broadly neutralizing antibody PGT121 allosterically modulates CD4    binding via recognition of the HIV-1 gp120 V3 base and multiple    surrounding glycans. PLoS Pathog 9, e1003342-   62. Depetris, R. S., Julien, J. P., Khayat, R., Lee, J. H., Pejchal,    R., Katpally, U., Cocco, N., Kachare, M., Massi, E., David, K. B.,    Cupo, A., Marozsan, A. J., Olson, W. C., Ward, A. B., Wilson, I. A.,    Sanders, R. W., and Moore, J. P. (2012) Partial enzymatic    deglycosylation preserves the structure of cleaved recombinant HIV-1    envelope glycoprotein trimers. J Biol Chem 287, 24239-24254-   63. Guttman, M., Garcia, N. K., Cupo, A., Matsui, T., Julien, J. P.,    Sanders, R. W., Wilson, I. A., Moore, J. P., and Lee, K. K. (2014)    CD4-induced activation in a soluble HIV-1 Env trimer. Structure 22,    974-984-   64. Sanders, R. W., Venturi, M., Schiffner, L., Kalyanaraman, R.,    Katinger, H., Lloyd, K. O., Kwong, P. D., and Moore, J. P. (2002)    The mannose-dependent epitope for neutralizing antibody 2G12 on    human immunodeficiency virus type 1 glycoprotein gp120. J Virol 76,    7293-7305-   65. Murin, C. D., Julien, J. P., Sok, D., Stanfield, R. L., Khayat,    R., Cupo, A., Moore, J. P., Burton, D. R., Wilson, I. A., and    Ward, A. B. (2014) Structure of 2G12 Fab2 in complex with soluble    and fully glycosylated HIV-1 Env by negative-stain single-particle    electron microscopy. J Virol 88, 10177-10188-   66. Yasmeen, A., Ringe, R., Derking, R., Cupo, A., Julien, J. P.,    Burton, D. R., Ward, A. B., Wilson, I. A., Sanders, R. W., Moore, J.    P., and Klasse, P. J. (2014) Differential binding of neutralizing    and non-neutralizing antibodies to native-like soluble HIV-1 Env    trimers, uncleaved Env proteins, and monomeric subunits.    Retrovirology 11, 41-   67. Pancera, M., and Wyatt, R. (2005) Selective recognition of    oligomeric HIV-1 primary isolate envelope glycoproteins by potently    neutralizing ligands requires efficient precursor cleavage. Virology    332, 145-156-   68. Blattner, C., Lee, J. H., Sliepen, K., Derking, R., Falkowska,    E., de la Pena, A. T., Cupo, A., Julien, J. P., van Gils, M.,    Lee, P. S., Peng, W., Paulson, J. C., Poignard, P., Burton, D. R.,    Moore, J. P., Sanders, R. W., Wilson, I. A., and Ward, A. B. (2014)    Structural delineation of a quaternary, cleavage-dependent epitope    at the gp41-gp120 interface on intact HIV-1 Env trimers. Immunity    40, 669-680-   69. Pancera, M., Zhou, T., Druz, A., Georgiev, I. S., Soto, C.,    Gorman, J., Huang, J., Acharya, P., Chuang, G. Y., Ofek, G.,    Stewart-Jones, G. B., Stuckey, J., Bailer, R. T., Joyce, M. G.,    Louder, M. K., Tumba, N., Yang, Y., Zhang, B., Cohen, M. S.,    Haynes, B. F., Mascola, J. R., Morris, L., Munro, J. B.,    Blanchard, S. C., Mothes, W., Connors, M., and Kwong, P. D. (2014)    Structure and immune recognition of trimeric pre-fusion HIV-1 Env.    Nature 514, 455-461-   70. McLellan, J. S., Pancera, M., Carrico, C., Gorman, J.,    Julien, J. P., Khayat, R., Louder, R., Pejchal, R., Sastry, M., Dai,    K., O'Dell, S., Patel, N., Shahzad-ul-Hussan, S., Yang, Y., Zhang,    B., Zhou, T., Zhu, J., Boyington, J. C., Chuang, G. Y., Diwanji, D.,    Georgiev, I., Kwon, Y. D., Lee, D., Louder, M. K., Moquin, S.,    Schmidt, S. D., Yang, Z. Y., Bonsignori, M., Crump, J. A.,    Kapiga, S. H., Sam, N. E., Haynes, B. F., Burton, D. R., Koff, W.    C., Walker, L. M., Phogat, S., Wyatt, R., Orwenyo, J., Wang, L. X.,    Arthos, J., Bewley, C. A., Mascola, J. R., Nabel, G. J., Schief, W.    R., Ward, A. B., Wilson, I. A., and Kwong, P. D. (2011) Structure of    HIV-1 gp120 V1N2 domain with broadly neutralizing antibody PG9.    Nature 480, 336-343-   71. Julien, J. P., Lee, J. H., Cupo, A., Murin, C. D., Derking, R.,    Hoffenberg, S., Caulfield, M. J., King, C. R., Marozsan, A. J.,    Klasse, P. J., Sanders, R. W., Moore, J. P., Wilson, I. A., and    Ward, A. B. (2013) Asymmetric recognition of the HIV-1 trimer by    broadly neutralizing antibody PG9. Proc Natl Acad Sci USA 110,    4351-4356-   72. Thali, M., Furman, C., Ho, D. D., Robinson, J., Tilley, S.,    Pinter, A., and Sodroski, J. (1992) Discontinuous, conserved    neutralization epitopes overlapping the CD4-binding region of human    immunodeficiency virus type 1 gp120 envelope glycoprotein. J Virol    66, 5635-5641-   73. Chen, L., Kwon, Y. D., Zhou, T., Wu, X., O'Dell, S., Cavacini,    L., Hessell, A. J., Pancera, M., Tang, M., Xu, L., Yang, Z. Y.,    Zhang, M. Y., Arthos, J., Burton, D. R., Dimitrov, D. S., Nabel, G.    J., Posner, M. R., Sodroski, J., Wyatt, R., Mascola, J. R., and    Kwong, P. D. (2009) Structural basis of immune evasion at the site    of CD4 attachment on HIV-1 gp120. Science 326, 1123-1127-   74. Cavacini, L. A., Duval, M., Robinson, J., and    Posner, M. R. (2002) Interactions of human antibodies, epitope    exposure, antibody binding and neutralization of primary isolate    HIV-1 virions. AIDS 16, 2409-2417-   75. Pugach, P., Ozorowski, G., Cupo, A., Ringe, R., Yasmeen, A., de    Val, N., Derking, R., Kim, H. J., Korzun, J., Golabek, M., de Los    Reyes, K., Ketas, T. J., Julien, J. P., Burton, D. R., Wilson, I.    A., Sanders, R. W., Klasse, P. J., Ward, A. B., and    Moore, J. P. (2015) A native-like SOSIP.664 trimer based on a HIV-1    subtype B env gene. J Virol-   76. Checkley, M. A., Luttge, B. G., and Freed, E. O. (2011) HIV-1    envelope glycoprotein biosynthesis, trafficking, and incorporation.    J Mol Biol 410, 582-608-   77. Sun, Z. Y., Oh, K. J., Kim, M., Yu, J., Brusic, V., Song, L.,    Qiao, Z., Wang, J. H., Wagner, G., and Reinherz, E. L. (2008) HIV-1    broadly neutralizing antibody extracts its epitope from a kinked    gp41 ectodomain region on the viral membrane. Immunity 28, 52-63-   78. Bartesaghi, A., Merk, A., Borgnia, M. J., Milne, J. L., and    Subramaniam, S. (2013) Prefusion structure of trimeric HIV-1    envelope glycoprotein determined by cryo-electron microscopy. Nat    Struct Mol Biol 20, 1352-1357-   79. Harris, A., Borgnia, M. J., Shi, D., Bartesaghi, A., He, H.,    Pejchal, R., Kang, Y. K., Depetris, R., Marozsan, A. J., Sanders, R.    W., Klasse, P. J., Milne, J. L., Wilson, I. A., Olson, W. C.,    Moore, J. P., and Subramaniam, S. (2011) Trimeric HIV-1 glycoprotein    gp140 immunogens and native HIV-1 envelope glycoproteins display the    same closed and open quaternary molecular architectures. Proc Natl    Acad Sci USA 108, 11440-11445

All documents, patents, journal articles and other materials cited inthe present application are incorporated herein by reference.

While the present invention has been disclosed with references tocertain embodiments, numerous modification, alterations, and changes tothe described embodiments are possible without departing from the sphereand scope of the present invention, as defined in the appended claims.Accordingly, it is intended that the present invention not be limited tothe described embodiments, but that it has the full scope defined by thelanguage of the following claims, and equivalents thereof.

What is claimed is:
 1. A recombinant protein comprising: a recombinantHIV-1 gp140, a linker, and a tag, wherein the tag is attached toC-terminus of the recombinant HIV-1 gp140 through the linker, whereinthe recombinant protein assembles into recombinant trimers in a culturemedium when the recombinant protein is produced by cells growing in theculture medium, wherein each of the recombinant trimers comprises arecombinant trimer base formed by the recombinant HIV-1 gp140 notincluding the linker and the tag, wherein the recombinant trimer basemimics a native HIV-1 envelope trimer, wherein the linker is notrequired for the formation of the recombinant trimer base, wherein thelinker allows the formation of the recombinant trimer base that mimics anative HIV-1 envelope trimer, wherein the linker has a sufficient lengthto separate the tag from the recombinant trimer base so that a bindingmolecule bound on a solid matrix is accessible to the tag without sterichindrance; and wherein the linker comprises at least 20 amino acids. 2.The recombinant protein of claim 1, wherein the linker comprises atleast 23 amino acids.
 3. The recombinant protein of claim 1, wherein thelinker comprises SEQ ID NO:
 1. 4. The recombinant protein of claim 1,wherein the linker comprises SEQ ID NO:
 5. 5. The recombinant protein ofclaim 1, wherein the linker comprises SEQ ID NO:
 6. 6. The recombinantprotein of claim 1, wherein the linker comprises SEQ ID NO:
 7. 7. Therecombinant protein of claim 1, wherein the linker comprises a flexiblelinker.
 8. The recombinant protein of claim 7, wherein the flexiblelinker comprises SEQ ID NO:
 8. 9. The recombinant protein of claim 1,wherein the linker comprises a rigid linker.
 10. The recombinant proteinof claim 9, wherein the rigid linker comprises SEQ ID NO:
 9. 11. Therecombinant protein of claim 1, wherein the tag comprises SEQ ID NO: 2.12. The recombinant protein of claim 1, wherein the tag comprises SEQ IDNO:
 3. 13. The recombinant protein of claim 1, wherein the recombinantHIV-1 gp140 comprises a gp120 and a gp41 ectodomain truncated at aa664based on HXB2 numbering, wherein the gp120 and the gp41 ectodomain arejoined by a junction sequence comprising a furin cleavage site REKR. 14.The recombinant protein of claim 13, wherein the furin cleavage siteREKR is mutated to SEKS, and wherein the recombinant HIV-1 gp140 isfurin cleavage resistant.
 15. The recombinant protein of claim 13,wherein the furin cleavage site REKR is mutated to SEQ ID NO: 13, andwherein the recombinant HIV-1 gp140 is furin cleavage proficient. 16.The recombinant protein of claim 1, wherein the recombinant HIV-1 gp140comprises “SOSIP” mutations that comprise A501C, T605C, and I559P, andwherein the recombinant HIV-1 gp140 comprises five “stabilizing”mutations comprising I535M, Q543L, S553N, K567Q, and R588G.
 17. Therecombinant protein of claim 1, where an HIV-1 clade B gp140 is used asa template to construct the recombinant HIV-1 gp140.
 18. The recombinantprotein of claim 1, where an HIV-1 clade A gp140 is used as a templateto construct the recombinant HIV-1 gp140.
 19. The recombinant protein ofclaim 1, wherein the recombinant protein comprises SEQ ID NO:
 10. 20.The recombinant protein of claim 1, wherein the recombinant proteincomprises SEQ ID NO:
 11. 21. The recombinant protein of claim 1, whereinthe recombinant protein comprises SEQ ID NO:
 12. 22. A compositioncomprising a recombinant trimer of a recombinant protein, wherein therecombinant protein comprises a recombinant HIV-1 gp140 attached to atag through a linker at C-terminus of the recombinant HIV-1 gp140,wherein the recombinant trimer comprises a recombinant trimer baseformed by the recombinant HIV-1 gp140 not including the linker and thetag, wherein the recombinant trimer base mimics a native HIV-1 envelopetrimer, wherein the linker is not required for the formation of therecombinant trimer base, wherein the linker allows the formation of therecombinant trimer base that mimics a native HIV-1 envelope trimer,wherein the linker has a sufficient length to separate the tag from therecombinant trimer base so that a binding molecule bound on a solidmatrix is accessible to the tag without steric hindrance; and whereinthe linker comprises at least 20 amino acids.
 23. The composition ofclaim 22, wherein the linker comprises at least 23 amino acids.
 24. Thecomposition of claim 22, wherein the linker comprises SEQ ID NO:
 1. 25.The composition of claim 22, wherein the linker comprises SEQ ID NO: 5.26. The composition of claim 22, wherein the linker comprises SEQ ID NO:6.
 27. The composition of claim 22, wherein the linker comprises SEQ IDNO:
 7. 28. The composition of claim 22, wherein the linker comprises aflexible linker.
 29. The composition of claim 28, wherein the flexiblelinker comprises SEQ ID NO:
 8. 30. The composition of claim 22, whereinthe linker comprises a rigid linker.
 31. The composition of claim 30,wherein the rigid linker comprises SEQ ID NO:
 9. 32. The composition ofclaim 22, wherein the tag comprises SEQ ID NO:
 2. 33. The composition ofclaim 22, wherein the tag comprises SEQ ID NO:
 3. 34. The composition ofclaim 22, wherein the recombinant HIV-1 gp140 comprises a gp120 and agp41 ectodomain truncated at aa664 based on HXB2 numbering, wherein thegp120 and the gp41 ectodomain are joined by a junction sequence thatcomprises a furin cleavage resistant sequence SEKS, and wherein therecombinant protein is furin cleavage resistant.
 35. The composition ofclaim 22, wherein the recombinant HIV-1 gp140 comprises “SOSIP”mutations that comprise A501C, T605C, and I559P, and wherein therecombinant HIV-1 gp140 comprises five “stabilizing” mutationscomprising I535M, Q543L, S553N, K567Q, and R588G.